阅读说明：本技术 用于蛋白质检测的组合物以及方法 (Compositions and methods for protein detection ) 是由 S·扬 R·塞斯勒 R·吉尔宝德 M·席尔姆 M·伊莎贝尔 G·格拉斯 于 2019-08-14 设计创作，主要内容包括：本发明总体上涉及具有特定电离特性的肽生物标记物,以通过液相色谱与串联质谱联用型多反应监测(MRM)直接定量包括转基因植物样品的生物样品中的一种或多种转基因靶蛋白。所述肽生物标记物与基于MRM的方法的组合可单独或组合使用选择的多种肽生物标记物用于定量堆叠的转基因作物诸如玉米中的单一转基因靶蛋白或多种转基因靶蛋白。本披露允许在包括植物基质的不同生物基质中进行广泛、可靠的定量。本发明的肽生物标记物可以进一步用作性状生物标记物以支持特定转基因事件的鉴定和/或选择。还提供了可用于执行本发明的方法的不同肽生物标记物组合。(The present invention relates generally to peptide biomarkers with specific ionization characteristics for direct quantification of one or more transgenic target proteins in a biological sample including a transgenic plant sample by liquid chromatography coupled with tandem mass spectrometry type Multiple Reaction Monitoring (MRM). The combination of the peptide biomarkers with MRM-based methods can use a selected plurality of peptide biomarkers, alone or in combination, for quantifying a single transgenic target protein or a plurality of transgenic target proteins in stacked transgenic crops, such as corn. The present disclosure allows for broad, reliable quantitation in different biological matrices, including plant matrices. The peptide biomarkers of the invention may further be used as trait biomarkers to support the identification and/or selection of specific transgenic events. Also provided are different peptide biomarker combinations useful in performing the methods of the invention.)

1. A labeled surrogate peptide that functions in a mass spectrometry assay to selectively detect or quantify a target transgenic protein selected from the group consisting of: a Cry1Ab protein, an ecry3.1ab protein, a mCry3A protein, a Vip3 protein, a double mutant 5-enolpyruvylshikimate-3-phosphate synthase (dmesps) protein, a glufosinate acetyltransferase (PAT) protein, and a phosphomannose isomerase (PMI) protein in a mixture of transgenic and non-transgenic proteins in one or more biological samples from one or more transgenic plants, said substitution peptide comprising a marker and an amino acid sequence selected from the group consisting of: GSAQGIEGSIR (SEQ ID NO:1), IVAQLGQGVYR (SEQ ID NO:2), TLSSTLYR (SEQ ID NO:3), DVSVFGQR (SEQ ID NO:4), TYPR (SEQ ID NO:5), TVSQLTR (SEQ ID NO:6), WYNTGTGLER (SEQ ID NO:7), EWEADPTNPALR (SEQ ID NO:8), VWPGPDSR (SEQ ID NO:9), APMFSWIHR (SEQ ID NO:10), WGFDAATINSR (SEQ ID NO:11), NQASSR (SEQ ID NO:12), IEEFAR (SEQ ID NO:13), SGFSNSSVSIIR (SEQ ID NO:14), LSHVSMFR (SEQ ID NO:15), 23 73 (SEQ ID NO:16), LEGLSNLYQIYAESFR (SEQ ID NO:17), YNFR (SEQ ID NO:18), YNDTRR (SEQ ID NO:19), SPHLMDILNSITIYTDAHR (SEQ ID NO:20), SAEFNNIIPSSQITQIPLTK (SEQ ID NO:21), QGFR (SEQ ID NO:22), SEQ ID NO: MDNNPNINECIPYNCLSNPEVEVLGGER (SEQ ID NO:24), SEQ ID NO:24), RPFNIGINNQQLSVLDGTEFAYGTSSNLPSAVYR (SEQ ID NO:25), SGTVDSLDEIPPQNNNVPPR (SEQ ID NO:26), TDVTDYHIDQV (SEQ ID NO:27), AVNELFTSSNQIGLK (SEQ ID NO:28), ITQLPLTK (SEQ ID NO:29), GLDSTTK (SEQ ID NO:30), QCAGIRPYDGR (SEQ ID NO:31), IEFVPAEVTFEAEYDLER (SEQ ID NO:32), ITQLPLVK (SEQ ID NO:33), MTADNNTEALDSSTTK (SEQ ID NO:34), VYIDK (SEQ ID NO:35), DGGISQFIGDK (SEQ ID NO:36), LITTLTCK (SEQ ID NO:37), ELLLATDLSNK (SEQ ID NO:38), FEELTFATETSSK (SEQ ID NO:39), EVLFEK (SEQ ID NO:40), TASELITK (SEQ ID NO:41), DVSEMFTTK (SEQ ID NO:42), LLGLADIDYTSIMNEHLNK (SEQ ID NO:43), IDFTK (SEQ ID NO:44), TDTGGDLTLDEILK (SEQ ID NO:45), DIMNK (SEQ ID NO:46), VHK (SEQ ID NO: 685), SEQ ID NO:47), and 68548, ITSMLSDVIK (SEQ ID NO:49), QNLQLDSFSTYR (SEQ ID NO:50), DSLSEVIYGDMDK (SEQ ID NO:51), MIVEAKPGHALIGFEISNDSITVLK (SEQ ID NO:52), VYFSVSGDANVR (SEQ ID NO:53), NQQLLNDISGK (SEQ ID NO:54), VESSEAEYR (SEQ ID NO:55), YMSGAK (SEQ ID NO:56), DGSPADILDELTELTELAK (SEQ ID NO:57), VYEAK (SEQ ID NO:58), LDAINTMLR (SEQ ID NO:59), GKPSIHLK (SEQ ID NO:60), DENTGYIHYEDTNNNLEDYQTINK (SEQ ID NO:61), DNFYIELSQGNNLYGGPIVHFYDVSIK (SEQ ID NO:62), LLCPDQSEQIYYTNNIVFPNEYVITK (SEQ ID NO:63), SQNGDEAWGDNFIILEISPSEK (SEQ ID NO:64), NAYVDHTGGVNGTK (SEQ ID NO:65), LDGVNGSLNDLIAQGNLNTELSK (SEQ ID NO:66), IANEQNQVLNDVNNK (SEQ ID NO:67), YEVTANFYDSSTGEIDLNK (SEQ ID NO:68), QNYALSLQIEYLSK (SEQ ID NO:69), QELISDK (SEQ ID NO:70), LLSPELINTNNWTSTGSTNISGNTLTLYQGGR (SEQ ID NO: 3671), and EK NO:72 (VN ID NO:72), QNYQVDK (SEQ ID NO:73), MAGAEEIVLQPIK (SEQ ID NO:74), FPVEDAK (SEQ ID NO:75), EISGTVK (SEQ ID NO:76), ILLLAALSEGTTVVDNLLNSEDVHYMLGALR (SEQ ID NO:77), DFELPAPPRPVRPVTQI (SEQ ID NO:78), LGLGSTLYTHLLK (SEQ ID NO:79), MSPER (SEQ ID NO:80), HGGWHDVGFWQR (SEQ ID NO:81), NAYDWTVESTVYVSHR (SEQ ID NO:82), TEPQTPQEWIDDLER (SEQ ID NO:83), AAGYK (SEQ ID NO:84), YPWLVAEVEGVVAGIAYAGPWK (SEQ ID NO:85), RPVEIRPATAADMAAVCDIVNHYIETSTVNFR (SEQ ID NO:86), ENAAGIPMDAAER (SEQ ID NO:87), ALAILK (SEQ ID NO:88), SALDSQQGEPWQTIR (SEQ ID NO:89), GSQQLQLKPGESAFIAANESPVTVK (SEQ ID NO:90), FEAKPANQLLTQPVK (SEQ ID NO:91), STLLGEAVAK (SEQ ID NO:92), LINSVQNYAWGSK (SEQ ID NO:93), HNSEIGFAK (SEQ ID NO:94), SEQ ID NO: VLCAAQPLSIQVHPNK (SEQ ID NO:95), TALTELYGMENPSSQPMAELWMGAHPK (SEQ ID NO:96), LSELFASLLNMQGEEK (SEQ ID NO:97) and QGAELDFPIPVDDFAFSLHDLSDK (SEQ ID NO: 98).

2. The labeled surrogate peptide of claim 1, wherein the peptide is labeled by incorporating a Stable Isotopically Labeled (SIL) amino acid.

3. The labeled replacement peptide of claim 2, wherein the SIL amino acid is lysine, isoleucine, valine, or arginine.

4. The labeled replacement peptide of claim 1, wherein the peptide selectively detects or quantifies the Cry1Ab protein in the mixture and comprises an amino acid sequence selected from the group consisting of seq id nos: GSAQGIEGSIR (SEQ ID NO:1), IVAQLGQGVYR (SEQ ID NO:2), TLSSTLYR (SEQ ID NO:3), DVSVFGQR (SEQ ID NO:4), TYPR (SEQ ID NO:5), TVSQLTR (SEQ ID NO:6), WYNTGTGLER (SEQ ID NO:7), EWEADPTNPALR (SEQ ID NO:8), VWPGPDSR (SEQ ID NO:9), APMFSWIHR (SEQ ID NO:10), WGFDAATINSR (SEQ ID NO:11), NQASSR (SEQ ID NO:12), IEEFAR (SEQ ID NO:13), SGFSNSSVSIIR (SEQ ID NO:14), LSHVSMFR (SEQ ID NO:15), 23 73 (SEQ ID NO:16), LEGLSNLYQIYAESFR (SEQ ID NO:17), YNFR (SEQ ID NO:18), YNDTRR (SEQ ID NO:19), SPHLMDILNSITIYTDAHR (SEQ ID NO:20), SAEFNNIIPSSQITQIPLTK (SEQ ID NO:21), QGFR (SEQ ID NO:22), SEQ ID NO: MDNNPNINECIPYNCLSNPEVEVLGGER (SEQ ID NO:24), SEQ ID NO:24), RPFNIGINNQQLSVLDGTEFAYGTSSNLPSAVYR (SEQ ID NO:25) and SGTVDSLDEIPPQNNNVPPR (SEQ ID NO: 26).

5. The surrogate peptide of claim 4, wherein the peptide produces a transition ion having an amino acid sequence selected from the group consisting of: GIEGSIR (SEQ ID NO:99), EGSIR (SEQ ID NO:100), AQLGQGVYR (SEQ ID NO:101), GQGVGR (SEQ ID NO:102), SSLYR (SEQ ID NO:103), STLYR (SEQ ID NO:104), SVFGQR (SEQ ID NO:105), FGQR (SEQ ID NO:106), PIR, TY, SQLTR (SEQ ID NO:107), QLTR (SEQ ID NO:108), NTGLER (SEQ ID NO:109), TGYNLER (SEQ ID NO:110), PTNPALR (SEQ ID NO:111), DPTNPALR (SEQ ID NO:112), GPDSR (SEQ ID NO:113), VW, HR, SWIHR (SEQ ID NO:114), ATINSR (SEQ ID NO:115), DANSATIR (SEQ ID NO:116), AISR (SEQ ID NO:117), ISR, EFID NO (SEQ ID NO:118), FAAR (SEQ ID NO:119), EEVSIIR (SEQ ID NO:120), and ESIIR (SEQ ID NO:121), SMFR (SEQ ID NO:122), VSMFR (SEQ ID NO:123), ENFDGSFR (SEQ ID NO:124), GSFR (SEQ ID NO:125), YAESFR (SEQ ID NO:126), LEG, NQFR (SEQ ID NO:127), QFR, DLTR (SEQ ID NO:128), NDLTR (SEQ ID NO:129), TIYTDAHR (SEQ ID NO:130), YTDAHR (SEQ ID NO:131), PLTK (SEQ ID NO:132), SAEFNNII (SEQ ID NO:133), FSHR (SEQ ID NO:134), GFSHR (SEQ ID NO:135), EVLGGER (SEQ ID NO:136), GGER (SEQ ID NO:137), NYFPDSR (SEQ ID NO:138), PNYDSR (SEQ ID NO:139), PSAVYR (SEQ ID NO:140), YR, PPR and SGTVDSLDE (SEQ ID NO: 141).

6. The replacement peptide of claim 4, wherein the peptide comprises amino acid sequence SAEFNNIIPSSQITQIPLTK (SEQ ID NO:21) and produces a transition ion consisting of the amino acid sequence PLTK (SEQ ID NO:132) or SAEFNNII (SEQ ID NO: 133).

7. The replacement peptide of claim 1, wherein the peptide selectively detects or quantifies an ecry3.1ab protein and comprises an amino acid sequence selected from the group consisting of: TDVTDYHIDQV (SEQ ID NO:27), AVNELFTSSNQIGLK (SEQ ID NO:28), ITQLPLTK (SEQ ID NO:29), GLDSTTK (SEQ ID NO:30), QCAGIRPYDGR (SEQ ID NO:31) and IEFVPAEVTFEAEYDLER (SEQ ID NO: 32).

8. The surrogate peptide of claim 7, wherein the peptide produces a transition ion having an amino acid sequence selected from the group consisting of: TDYHIDQV (SEQ ID NO:142), DYYHIDQV (SEQ ID NO:143), TSSNQIGLK (SEQ ID NO:144), SSNQIGLK (SEQ ID NO:145), QLPLTK (SEQ ID NO:146), TQLPLTK (SEQ ID NO:147), DSTTK (SEQ ID NO:148), SSTTK (SEQ ID NO:149), PYDGR (SEQ ID NO:150), DGR, IEF and LER.

9. The surrogate peptide of claim 7, wherein the peptide comprises amino acid sequence TDVTDYHIDQV (SEQ ID NO:27) and produces a transition ion consisting of amino acid sequence TDYHIDQV (SEQ ID NO:142) or DYHIDQV (SEQ ID NO: 143).

10. The replacement peptide of claim 1, wherein the peptide selectively detects or quantifies mCry3A protein and comprises an amino acid sequence selected from the group consisting of: ITQLPLVK (SEQ ID NO:33), MTADNNTEALDSSTTK (SEQ ID NO:34) and VYIDK (SEQ ID NO: 35).

11. The surrogate peptide of claim 10, wherein the peptide produces a transition ion having an amino acid sequence selected from the group consisting of: QLPLVK (SEQ ID NO:151), TQLPLVK (SEQ ID NO:152), ALDSTTK (SEQ ID NO:153), EALDSSTTK (SEQ ID NO:154), YIDK (SEQ ID NO:155), and IDK.

12. The replacement peptide of claim 1, wherein the peptide selectively detects or quantifies Vip3A protein and comprises an amino acid sequence selected from the group consisting of: DGGISQFIGDK (SEQ ID NO:36), LITTLTCK (SEQ ID NO:37), ELLLATDLSNK (SEQ ID NO:38), FEELTFATETSSK (SEQ ID NO:39), EVLFEK (SEQ ID NO:40), TASELITK (SEQ ID NO:41), DVSEMFTTK (SEQ ID NO:42), LLGLADIDYTSIMNEHLNK (SEQ ID NO:43), IDFTK (SEQ ID NO:44), TDTGGDLTLDEILK (SEQ ID NO:45), DIMNMIFK (SEQ ID NO:46), ALYVHK (SEQ ID NO:47), VNILPTLSNTFSNPNYAK (SEQ ID NO:48), ITSMLSDVIK (SEQ ID NO:49), QNLQLDSFSTYR (SEQ ID NO:50), DSLSEVIYGDMDK (SEQ ID NO:51), MIVEAKPGHALIGFEISNDSITVLK (SEQ ID NO:52), VYFSVSGDANVR (SEQ ID NO:53), NQQLLNDISGK (SEQ ID NO:54), VESSEAEYR (SEQ ID NO:55), YMAK (SEQ ID NO:56), DGSPADILDELTELTELAK (SEQ ID NO:57), YE NO:36), and 8959 (SEQ ID NO: 8958), GKPSIHLK (SEQ ID NO:60), DENTGYIHYEDTNNNLEDYQTINK (SEQ ID NO:61), DNFYIELSQGNNLYGGPIVHFYDVSIK (SEQ ID NO:62), LLCPDQSEQIYYTNNIVFPNEYVITK (SEQ ID NO:63), SQNGDEAWGDNFIILEISPSEK (SEQ ID NO:64), NAYVDHTGGVNGTK (SEQ ID NO:65), LDGVNGSLNDLIAQGNLNTELSK (SEQ ID NO:66), IANEQNQVLNDVNNK (SEQ ID NO:67), YEVTANFYDSSTGEIDLNK (SEQ ID NO:68), QNYALSLQIEYLSK (SEQ ID NO:69), QLQEIISDK (SEQ ID NO:70), LLSPELINTNNWTSTGSTNISGNTLTLYQGGR (SEQ ID NO:71), YVNEK (SEQ ID NO:72) and QNYQVDK (SEQ ID NO: 73).

13. The surrogate peptide of claim 12, wherein the peptide produces a transition ion having an amino acid sequence selected from the group consisting of: SQFIGDK (SEQ ID NO:156), GDK, TLTCK (SEQ ID NO:157), TCK, ATDLSNK (SEQ ID NO:158), LATDLSNK (SEQ ID NO:159), TFATETSSK (SEQ ID NO:160), FATETSSK (SEQ ID NO:161), FEK, LFEK (SEQ ID NO:162), SELITK (SEQ ID NO:163), ASELITK (SEQ ID NO:164), SEMFTTK (SEQ ID NO:165), DVS, IMNEHLNK (SEQ ID NO:166), MNLNK (SEQ ID NO:167), DFTK (SEQ ID NO:168), FTK, TLDEILK (SEQ ID NO:169), LTEILK (SEQ ID NO:170), MNMIFK (SEQ ID NO:171), NMIFK (SEQ ID NO:172), YVHK (SEQ ID NO:173), VNHK, SEQ ID NO:174), SMLSR (SEQ ID NO:176), SMLSR (SEQ ID NO:177), VNSTISK (SEQ ID NO:177), NMIFR (SEQ ID NO:178), NMIFNYS NO:177), and SEQ ID NO:177, IYGDMDK (SEQ ID NO:179), VIYGDMDK (SEQ ID NO:180), SNDSITVLK (SEQ ID NO:181), MIV, SGDANVR (SEQ ID NO:182), SVSGDANVR (SEQ ID NO:183), LLNDISGK (SEQ ID NO:184), LNDISGK (SEQ ID NO:185), SSEAEYR (SEQ ID NO:186), ESSEAEYR (SEQ ID NO:187), SGAK (SEQ ID NO:188), MSGAK (SEQ ID NO:189), TELTELAK (SEQ ID NO:190), DGSPADI (SEQ ID NO:191), YEAK (SEQ ID NO:192), EAK, NTMLR (SEQ ID NO:193), AINTR (SEQ ID NO:194), PSDNLK (SEQ ID NO:195), HLK, DYQTINK (SEQ ID NO:196), NK, SEF, FYFY (SEQ ID NO:197), PNEYMLTK (SEQ ID NO:198), PNE TK (SEQ ID NO:199), DHESSEYNKK, SEQ ID NO:200, DHESSENGITK (SEQ ID NO:199), DHISTK: 200, DHESSETK, DHESSETKI (SEQ ID NO:199), and DHISTK), GNLNTELSK (SEQ ID NO:202), NTELSK (SEQ ID NO:203), LNDVNNK (SEQ ID NO:204), NDVNNK (SEQ ID NO:205), YE, DLNK (SEQ ID NO:206), QIEYLSK (SEQ ID NO:207), LQIEYLSK (SEQ ID NO:208), SDK, QEISDK (SEQ ID NO:209), YQGGR (SEQ ID NO:210), TLYQGGR (SEQ ID NO:211), NEK, VNEK (SEQ ID NO:212), DK, and VDK.

14. The replacement peptide of claim 1, wherein the peptide selectively detects or quantifies a dmesps protein and comprises an amino acid sequence selected from the group consisting of: MAGAEEIVLQPIK (SEQ ID NO:74), FPVEDAK (SEQ ID NO:75), EISGTVK (SEQ ID NO:76), and ILLLAALSEGTTVVDNLLNSEDVHYMLGALR (SEQ ID NO: 77).

15. The surrogate peptide of claim 14, wherein the peptide produces a transition ion having an amino acid sequence selected from the group consisting of: PIK, EIVLQPIK (SEQ ID NO:213), PVEDAK (SEQ ID NO:214), VEDAK (SEQ ID NO:215), SGTVK (SEQ ID NO:216), GTVK (SEQ ID NO:217), ILLLAA (SEQ ID NO:218), and HYMLGALR (SEQ ID NO: 219).

16. The replacement peptide of claim 1, wherein the peptide selectively detects or quantifies the PAT protein and comprises an amino acid sequence selected from the group consisting of: DFELPAPPRPVRPVTQI (SEQ ID NO:78), LGLGSTLYTHLLK (SEQ ID NO:79), MSPER (SEQ ID NO:80), HGGWHDVGFWQR (SEQ ID NO:81), NAYDWTVESTVYVSHR (SEQ ID NO:82), TEPQTPQEWIDDLER (SEQ ID NO:83), AAGYK (SEQ ID NO:84), YPWLVAEVEGVVAGIAYAGPWK (SEQ ID NO:85) and RPVEIRPATAADMAAVCDIVNHYIETSTVNFR (SEQ ID NO: 86).

17. The surrogate peptide of claim 16, wherein the peptide produces a transition ion having an amino acid sequence selected from the group consisting of: DFE, DF, YTHLLK (SEQ ID NO:220), THLLK (SEQ ID NO:221), PER, SPER (SEQ ID NO:222), GFWQR (SEQ ID NO:223), VGFWQR (SEQ ID NO:224), STVYVSHR (SEQ ID NO:225), SHR, TEPQT (SEQ ID NO:226), DLER (SEQ ID NO:227), GYK, AGYK (SEQ ID NO:228), GPWK (SEQ ID NO:229), GIAYAGPWK (SEQ ID NO:230), TSTVNFR (SEQ ID NO:231), and NFR.

18. The surrogate peptide according to claim 16, wherein the peptide comprises the amino acid sequence LGLGSTLYTHLLK (SEQ ID NO:79) and generates a transition ion consisting of the amino acid sequence YTHLLK (SEQ ID NO:220) or THLLK (SEQ ID NO: 221).

19. The replacement peptide of claim 1, wherein the peptide selectively detects or quantifies PMI protein and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: ENAAGIPMDAAER (SEQ ID NO:87), ALAILK (SEQ ID NO:88), SALDSQQGEPWQTIR (SEQ ID NO:89), GSQQLQLKPGESAFIAANESPVTVK (SEQ ID NO:90), FEAKPANQLLTQPVK (SEQ ID NO:91), STLLGEAVAK (SEQ ID NO:92), LINSVQNYAWGSK (SEQ ID NO:93), HNSEIGFAK (SEQ ID NO:94), VLCAAQPLSIQVHPNK (SEQ ID NO:95), TALTELYGMENPSSQPMAELWMGAHPK (SEQ ID NO:96), LSELFASLLNMQGEEK (SEQ ID NO:97) and QGAELDFPIPVDDFAFSLHDLSDK (SEQ ID NO: 98).

20. The surrogate peptide of claim 19, wherein the peptide produces a transition ion having an amino acid sequence selected from the group consisting of: PMDAAER (SEQ ID NO:232), GIPMDAAER (SEQ ID NO:233), AILK (SEQ ID NO:234), LK, PWQTIR (SEQ ID NO:235), GEPWQTIR (SEQ ID NO:236), ANESPVTVK (SEQ ID NO:237), PVTVK (SEQ ID NO:238), LTQPVK (SEQ ID NO:239), PVK, GEAVAK (SEQ ID NO:240), LGEAVAK (SEQ ID NO:241), QNYAWGSK (SEQ ID NO:242), NYAWGSK (SEQ ID NO:243), NSEIGFAK (SEQ ID NO:244), HN, VLCAAQ (SEQ ID NO:245), PNK, WMGAHPK (SEQ ID NO:246), TALTE (SEQ ID NO:247), NMQGEEK (SEQ ID NO:248), LNMQGEEK (SEQ ID NO:249), SLHDLSDK (SEQ ID NO:250), and SEQ ID NO: 251).

21. The surrogate peptide of claim 19, wherein the peptide comprises amino acid sequence SALDSQQGEPWQTIR (SEQ ID NO:89) and produces a transition ion consisting of amino acid sequence PWQTIR (SEQ ID NO:235) or GEPWQTIR (SEQ ID NO: 236).

22. The replacement peptide of claim 4, wherein the Cry1Ab protein comprises the amino acid sequence of SEQ ID NO 259.

23. The replacement peptide of claim 22, wherein the Cry1Ab protein is from event Bt 11.

24. The replacement peptide of claim 7, wherein the ecry3.1ab protein comprises the amino acid sequence of SEQ ID NO: 260.

25. The replacement peptide of claim 24, wherein the ecry3.1ab protein is from event 5307.

26. The replacement peptide of claim 10, wherein the mCry3A protein comprises the amino acid sequence of SEQ ID No. 261.

27. The replacement peptide of claim 26, wherein the mCry3A protein is from event MIR 604.

28. The replacement peptide of claim 12, wherein the Vip3A protein comprises the amino acid sequence of SEQ ID NO 262.

29. The replacement peptide of claim 28, wherein the Vip3A protein is from event MIR 162.

30. The replacement peptide of claim 14, wherein the dmesps protein comprises the amino acid sequence of SEQ ID NO: 263.

31. The replacement peptide of claim 30, wherein the dmesps protein is from event GA 21.

32. The replacement peptide of claim 16, wherein the PAT protein comprises the amino acid sequence of SEQ ID NO 264.

33. The surrogate peptide of claim 32, wherein the PAT protein is from event Bt11, 59122, TC1507, DP4114, or T25.

34. The replacement peptide of claim 19, wherein the PMI protein comprises the amino acid sequence of SEQ ID NO 265 or SEQ ID NO 266.

35. The replacement peptide of claim 34, wherein the PMI protein is from event MIR162, event MIR604, event 5307, or event 3272.

36. The replacement peptide of any one of claims 1-35, wherein the mixture of transgenic proteins comprises at least two transgenic proteins selected from the group consisting of: cry1Ab protein, ecry3.1ab protein, mCry3A protein, Vip3A protein, dmesps protein, PAT protein, and PMI protein.

37. The labeled replacement peptide of claim 36, wherein the mixture of transgenic proteins comprises a Cry1Ab protein, an ecry3.1ab protein, an mCry3A protein, a Vip3A protein, a dmesps protein, a PAT protein, and a PMI protein.

38. The labeled replacement peptide of claim 37, wherein the mixture of transgenic proteins further comprises at least one transgenic protein selected from the group consisting of: Cry1A.105 protein (SEQ ID NO:267), Cry2Ab protein (SEQ ID NO:268), Cry1F protein (SEQ ID NO:269), Cry34 protein (SEQ ID NO:270), and Cry35 protein (SEQ ID NO: 271).

39. The replacement peptide of claim 1, wherein the transgenic plant is selected from the group consisting of: corn, soybean, cotton, rice, wheat, canola, and eggplant.

40. The replacement peptide of claim 39, wherein the transgenic corn plant comprises a transgenic corn event selected from the group consisting of: event Bt11, event 5307, event MIR604, and event GA 21.

41. The replacement peptide of claim 40, wherein the transgenic corn plant comprises event Bt11, event 5307, MIR604, and event GA 21.

42. The replacement peptide of claim 41, wherein the transgenic corn plant further comprises event 3272, event MON89034, event DP4114, event 1577, or event 59122.

43. The replacement peptide of claim 1, wherein the biological sample is from leaf tissue, seeds, grain, pollen, or root tissue.

44. The surrogate peptide of claim 43, wherein the biological sample comprises a Cry1Ab protein from event Bt11, an eCry3.1Ab protein from event 5307, an mCry3A protein from event MIR604, an EPSPS protein from event GA21, a PAT protein from Bt11, 59122, DP4114, TC1507 or T25, or a PMI protein from event MIR162, event MIR604, event 5307 or event 3272.

45. The surrogate peptide of claim 44, wherein the biological sample further comprises a Cry1A.105 protein from event MON89034, a Cry1F protein from event 1507, or Cry34 and Cry35 proteins from event 59122.

46. An assay kit comprising at least two labeled surrogate peptides according to claim 1.

47. A method of simultaneously detecting or quantifying one or more target transgenic proteins in a complex biological sample comprising a mixture of the target transgenic protein and a non-transgenic protein from a transgenic plant, the method comprising:

a. obtaining a biological sample from the transgenic plant;

b. extracting proteins from said biological sample to obtain an extract comprising a mixture of proteins;

c. reducing the amount of non-transgenic insoluble protein in the extract of step b to obtain a concentrated extract of soluble protein;

d. digesting the soluble proteins in the extract of step c to obtain an extract comprising peptide fragments, wherein the peptide fragments comprise at least one surrogate peptide specific for each target transgenic protein;

e. concentrating the peptide fragments of the extract of step d,

f. adding one or more labeled replacement peptides of claim 1, wherein each labeled replacement peptide has the same amino acid sequence as each replacement peptide of the target transgenic protein, and wherein the number of labeled replacement peptides added is equal to the number of target transgenic proteins in the mixture;

g. concentrating the replacement peptide and the labeled replacement peptide by reducing the amount of non-replacement peptide in the mixture;

h. decomposing the peptide fragment mixture of step g by liquid chromatography;

i. analyzing the peptide fragment mixture from step h by mass spectrometry, wherein detection of a transition ion fragment of the labeled surrogate peptide indicates the presence of the target transgenic protein from which the surrogate peptide was derived; and, optionally,

j. calculating the amount of the target transgenic protein in the biological sample by comparing the mass spectral signal generated by the transition ion fragment of step i with the mass spectral signal generated by the transition ion of the labeled surrogate peptide.

48. The method of claim 47, wherein said target transgenic protein is a Cry1Ab protein, an eCry3.1Ab protein, an mCry3A protein, a Vip3 protein, a double mutant 5-enolpyruvylshikimate-3-phosphate synthase (dmePSPS) protein, a glufosinate acetyltransferase (PAT) protein, or a phosphomannose isomerase (PMI) protein.

49. The method of claim 48, wherein the target transgenic protein is Cry1Ab and the labeled replacement peptide comprises amino acid sequence SAEFNNIIPSSQITQIPLTK (SEQ ID NO:21) and produces a transition ion consisting of the amino acid sequence PLTK (SEQ ID NO:132) or SAEFNNII (SEQ ID NO: 133).

50. The method of claim 49, wherein the Cry1Ab target protein in the biological sample is quantified by comparing mass spectral signals generated by transition ion fragments consisting of the amino acid sequence PLTK (SEQ ID NO: 132).

51. The method of claim 47, wherein the target transgenic protein is an eCry3.1Ab protein and the tagged replacement peptide comprises amino acid sequence TDVTDYHIDQV (SEQ ID NO:27) and generates a transition ion consisting of the amino acid sequence TDYHIDQV (SEQ ID NO:142) or DYYHIDQV (SEQ ID NO: 143).

52. The method of claim 51, wherein the eCry3.1Ab transgene protein in the biological sample is quantified by comparing mass spectral signals generated by transition ion fragments consisting of amino acid sequences.

53. The method of claim 38, wherein the labeled replacement peptide comprises amino acid sequence DGGISQFIGDK (SEQ ID NO:36) and produces a transition ion consisting of amino acid sequence SQFIGDK (SEQ ID NO:156) or GDK.

54. The method of claim 38, wherein the labeled replacement peptide comprises amino acid sequence LGLGSTLYTHLLK (SEQ ID NO:79) and produces a transition ion consisting of the amino acid sequence YTHLLK (SEQ ID NO:220) or THLLK (SEQ ID NO: 221).

55. The method of claim 38, wherein the labeled replacement peptide comprises amino acid sequence SALDSQQGEPWQTIR (SEQ ID NO:89) and generates a transition ion consisting of the amino acid sequence PWQTIR (SEQ ID NO:235) or GEPWQTIR (SEQ ID NO: 236).

Technical Field

The present invention relates generally to the use of mass spectrometry for the selective detection, quantification and characterization of target transgenic proteins in complex biological samples.

Background

Transgenic crops consist of increasingly complex genetic modifications (including multiple transgenes conferring different traits, also known as "gene stacking" or "trait stacking"). For example, many transgenic corn products currently on the market contain within the same plant a variety of insecticidal proteins for controlling a broad spectrum of insect pests, a variety of proteins that confer tolerance to a broad spectrum of chemical herbicides on plants, and a variety of proteins that are used as selectable markers in plant transformation processes. Many transgenic proteins used to control insect pests, such as crystal endotoxins from bacillus thuringiensis (known as Cry proteins), can be structurally closely related to each other and have similar overall amino acid sequence identity or contain motifs or domains with significant identity. Many Cry proteins are active against lepidopteran or coleopteran insect pests. Examples of lepidopteran active Cry proteins include Cry1A, Cry1B, Cry1C, Cry1D, Cry1E, Cry1F, and Cry 9. Examples of coleopteran-active Cry proteins include Cry3A, Cry3B, Cry3C, Cry8, binary Cry23-Cry37, and binary Cry34-Cry 35. Within a given insect order, most individual Cry proteins have biological activity against a narrow spectrum of insect species.

Numerous successful attempts to create hybrid Cry proteins with enhanced activity profiles have been disclosed in the literature. For example, a silkworm moth (Bombyx mori) specific domain from the Cry1Aa protein was moved to the Cry1Ac protein, thereby conferring a novel insecticidal activity to the resulting Cry1Aa-Cry1Ac chimeric protein (Ge et al 1989, PNAS [ Proc. Natl. Acad. Sci. USA ]86: 40374041). Thompson et al, 1996 and 1997 (U.S. Pat. Nos. 5,527,883 and 5,593,881) replaced the protoxin tail region of the wild-type Cry1F protein and Cry1C protein with the protoxin tail region of the Cry1Ab protein to make a Cry1F-Cry1Ab hybrid Cry protein and a Cry1C-Cry1Ab hybrid Cry protein, which all have improved expression in certain expression host cells. Bosch et al, 1998 (U.S. Pat. No. 5,736,131) created a novel lepidopteran active protein by: substitution of domain III of the Cry1Ea and Cry1Ab proteins with domain III of the Cry1Ca protein resulted in a Cry1E-Cry1C hybrid Cry protein designated G27 and a Cry1Ab-Cry1C hybrid Cry protein designated H04, both having a broader lepidopteran activity profile than the wild-type Cry protein parent molecule. Malvar et al, 2001 (U.S. patent 6,242,241) combines domain I of Cry1Ac protein with domains II and III of Cry1F protein and protoxin tails to produce a Cry1Ac-Cry1F hybrid Cry protein having broader insecticidal activity than the parent wild-type Cry protein. Bogdanova et al, 2011 (us patent 8,034,997) combines domains I and II of the Cry1Ab protein with domain III of the Cry1Fa protein and adds the Cry1Ac protoxin tail to produce a novel lepidopteran active hybrid Cry protein known as cry1a.105. Furthermore, Hart et al, 2012 (U.S. patent 8,309,516) combines domains I and II of the Cry3A protein and the modified Cry3A protein with domain III of the Cry1Ab protein and adds a portion of the protoxin tail of the Cry1Ab protein to produce a coleopteran-active hybrid Cry protein (also known as ecry3.1ab) known as FR8 a. Most hybrid Cry proteins reported to date have used all or part of the same type of wild-type Cry protein, such as Cry1Aa, Cry1Ab, Cry1Ac, Cry1C, Cry1F, and Cry3A.

Several wild-type Cry proteins (e.g., Cry1Ab, Cry1Ac, Cry1C, Cry1F, Cry2A, Cry2Ba, Cry3A, Cry3B, Cry9C, and Cry34-Cry35) and vegetative insecticidal proteins (e.g., Vip3A (see U.S. patent 5,877,012)) have been expressed in transgenic crop plants (including corn, cotton, rice, and soybean), some of which have been commercially used since 1996 to control certain lepidopteran and coleopteran insect pests. Recently, transgenic crop products, such as corn, containing engineered Cry proteins in which one or more amino acids are substituted, deleted or inserted (e.g., modified Cry3A (mCry 3A; US patent 7,230,167)) and hybrid Cry proteins (e.g., the above-mentioned eCry3.1Ab and Cry1A.105) have been introduced commercially.

Recombinant DNA technology is increasingly used to produce transgenic plants for commercial and industrial use, which requires the development of diagnostic methods for analyzing transgenic plant lines. Such methods are needed to maintain transgenic plant varieties through successive generations of breeding, monitor the environment or the presence of transgenic plants or plant parts in biological samples derived from transgenic plants, and assist in the rapid generation and development of new transgenic plants with desired or optimal phenotypes. Furthermore, current regulatory agencies in many countries require guidelines for safety assessment of transgenic plants at the DNA and protein levels in order to obtain and maintain regulatory approval. The increasing complexity of genes and proteins stacked into transgenic plants as described above makes it difficult to detect and quantify the specificity of any one target protein within a complex mixture, particularly if the stacked transgenic proteins are similar to each other, or to wild-type non-transgenic proteins in the environment, or to non-transgenic proteins endogenous to the transgenic plant.

Immunoassays, such as enzyme-linked immunosorbent assays (ELISA), are currently the preferred method used in the agricultural industry for the detection and quantification of proteins introduced by genetic modification of plants. A key component of immunoassays is the antibody specific for the target protein (antigen). Immunoassays can be highly specific and the sample usually requires only a simple preparation before it is analyzed. Furthermore, immunoassays can be used qualitatively or quantitatively over a wide range of concentrations. Typically, immunoassays require separate tests for each protein of interest. The antibody may be polyclonal as produced in an animal or monoclonal as produced by cell culture. By its nature, a mixture of polyclonal antibodies will have multiple recognition epitopes that may increase sensitivity, but may also decrease specificity, as the likelihood of sequence and structure homology to other proteins increases with the number of different antibody paratopes present. Monoclonal antibodies have some advantages over polyclonal antibodies because they express uniform affinity and specificity for a single epitope or antigenic determinant, and can be produced in large quantities. However, the presence of inherent properties in all antibodies limits their use in more demanding applications, such as selective detection and quantification of individual transgenic proteins in a complex mixture of similar transgenic or endogenous proteins. In addition, both polyclonal and monoclonal antibodies may require further purification steps to enhance sensitivity and reduce background in the assay. In addition, the ELISA system may not be able to detect subtle changes in the target protein, and these changes may have a dramatic effect on its physical and biological properties. For example, the antibody may not recognize a particular form of the protein or peptide that has been altered by post-translational modifications (such as phosphorylation or glycosylation) or that is conformationally obscured or modified by partial degradation. The identification of such modifications is crucial, as changes in the physical and biological properties of these proteins may play an important role in their enzymatic, clinical or other biological activities. Such variations may limit the reliability and utility of ELISA-based quantitative methods.

Currently, efficient identification or quantification of transgenic protein in commercial crop products from transgenic plant products containing the transgenic protein depends on the accuracy of the immunoassay. The development of a successful immunoassay depends on certain characteristics of the antigen used to develop the antibody, namely the size, hydrophobicity and tertiary structure of the antigen and the quality and accuracy of the antibody. The specificity of the antibody must be carefully examined to elucidate any cross-reactivity with similar substances that may lead to false positive results. A problem with the industry today is that many antibodies in commercially available test kits do not distinguish between similar transgenic proteins in various products, or between transgenic proteins and wild-type proteins, which makes differential product identification and quantification difficult or impossible. For example, in the case of many current commercial transgenic crop products that use one or more of the same wild-type Cry proteins (e.g., Cry1Ab, Cry1Ac, Cry1F, and Cry3), and in the case of the introduction of crops that express hybrid Cry proteins that consist of all or part of the same wild-type Cry protein already in the transgenic crop product, there remains a need to develop new and improved diagnostic methods to be able to distinguish the wild-type Cry proteins from each other when together in a complex biological sample (e.g., a sample from a transgenic plant, transgenic plant part, or transgenic microorganism) and to distinguish the wild-type Cry protein from a hybrid Cry protein containing all or part of the same wild-type Cry protein.

Mass Spectrometry (MS) provides an alternative platform that overcomes many of the limitations of ELISA for protein analysis. The field of MS-based analysis has led to significant advances in targeted protein analysis, such as Multiple Reaction Monitoring (MRM) by electrospray liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The basic concept is that proteins can be quantified by measuring their specific constituent peptides (surrogate peptides) after proteolytic digestion. Data acquisition only for selected peptides allows measurements to be made with greater accuracy, sensitivity and throughput. Protein quantification by MRM-based measurement of surrogate peptides is the fastest growing application of MS in protein analysis. Compared to immune-based assays, MRM-based protein assays have two compelling advantages, the first being the ability to systematically configure specific assays against essentially any protein without the use of antibodies. The second is the ability of the targeted MS assay to perform multiple assays on many peptides in a single assay. Furthermore, MRM is a direct assay, whereas immune-based assays are indirect. Immuno-based assays rely on binding assays that include a linking reagent that can be immobilized on a solid phase and a detection reagent that specifically binds and uses an enzyme to generate a signal that can be correctly quantified.

Commercial transgenic crop products comprise a stack of insecticidal proteins, herbicide tolerance proteins, and selectable marker proteins. In the case of many commercial transgenic crop products that use one or more of the same wild-type insecticidal Cry proteins (e.g., Cry1Ab, Cry1F, and Cry3), and in the case of crops that have been introduced that express hybrid Cry insecticidal proteins (e.g., mCry3A, ecry3.1ab, and cry1a.105) that consist of all or part of the same wild-type Cry proteins already present in the transgenic crop product, MRM-based assays must be able to distinguish between these closely related transgenic target insecticidal proteins as well as herbicide tolerance proteins and selectable marker proteins. Thus, there is a continuing need to identify surrogate peptides that have all the biochemical properties that play a role in MRM-based assays, and that have additional properties such that these surrogate peptides are absolutely specific for targeting transgenic proteins that may have mostly overlapping amino acid sequences, i.e., one or more transition states of the surrogate peptide can clearly, without interference, distinguish between two closely related target proteins in multiple complex matrices. Such selective replacement peptides and their transition states should be able to distinguish target transgenic proteins that are similar to each other, or similar to wild-type non-transgenic proteins in the environment, or similar to non-transgenic proteins endogenous to the transgenic plant.

Disclosure of Invention

The present invention provides labeled surrogate peptides and their respective transition ions that can be used to selectively detect or quantify target transgenic proteins in complex biological matrices using mass spectrometry. The invention further provides methods and systems for selectively detecting or quantifying a target transgenic protein in a complex biological matrix using a labeled surrogate peptide and a transition ion.

In one aspect of the invention, the internal standard peptide tags are designed by empirical analysis and in silico digestion analysis; and genetically synthesized by chemical synthesis with heavy amino acid residues or by expression of synthetic genes in the presence of one or more amino acids or metabolic intermediates that are stable isotopically labeled. In certain embodiments, the standard may be characterized separately by Mass Spectrometry (MS) analysis, including tandem mass spectrometry (MS/MS), more particularly, liquid chromatography in combination with tandem mass spectrometry (LC-MS/MS). After characterization, preselected parameters for the peptides, such as the monoisotopic mass of each peptide, its surrogate charge state, surrogate m/z values, m/z transition ions, and ion type for each transition ion, can be collected. Other considerations include optimizing peptide size, avoiding post-translational modifications, avoiding process-induced modifications, and avoiding high rates of protease cleavage misses.

Provided herein is an exemplary list of unique Stable Isotope Labeled (SIL) replacement peptides, including peptides comprising any one or combination of SEQ ID NOs 1-98, for selective detection or quantification of transgenic proteins selected from the group consisting of: insecticidal proteins Cry1Ab, ecry3.1ab, mCry3A and Vip3, herbicide tolerance proteins dmesps and PAT, and a plant transformation selectable marker protein PMI that may be included in plants having a single transgenic event, a breeding stack of multiple events, or a molecular stack of multiple target transgenic proteins. Each surrogate peptide sequence and transition ion derived from each peptide of the seven proteins can be used in mass spectrometry-based Multiple Reaction Monitoring (MRM) assays.

In another aspect of the invention, the labeled replacement peptide selectively detects or quantifies the Cry1Ab protein and comprises the amino acid sequence of any one of SEQ ID NOs 1-26. In another aspect, the labeled replacement peptide selectively detects or quantifies a Cry1Ab protein and produces a transition ion having an amino acid sequence selected from SEQ ID NOs 99-141 or at least one of the peptides PIR, TY, VW, HR, YR, or PPR. In one embodiment of this aspect, the labeled replacement peptide comprises amino acid sequence SAEFNNIIPSSQITQIPLTK (SEQ ID NO:21) and generates a transition ion consisting of the amino acid sequence PLTK (SEQ ID NO:132) or SAEFNNII (SEQ ID NO: 133).

In another aspect of the invention, the labeled replacement peptide selectively detects or quantifies eCry3.1Ab protein and comprises the amino acid sequence of any one of SEQ ID NOs 27-32. In another aspect of the invention, the labeled replacement peptide selectively detects or quantifies the eCry3.1Ab protein and generates a transition ion having an amino acid sequence selected from the group consisting of SEQ ID NO:142-150 or at least one of the peptides DGR, IEF or LER. In one embodiment of this aspect, the labeled replacement peptide comprises amino acid sequence TDVTDYHIDQV (SEQ ID NO:27) and generates a transition ion consisting of amino acid sequence TDYHIDQV (SEQ ID NO:142) or DYKIDQV (SEQ ID NO: 143).

In another aspect of the invention, the labeled replacement peptide selectively detects or quantifies the protein mCry3A and comprises the amino acid sequence of any one of SEQ ID NOS 33-35. In another aspect of the invention, the labeled replacement peptide selectively detects or quantifies the mCry3A protein and generates a transition ion having an amino acid sequence selected from at least one of SEQ ID NO 151-155 or the peptide IDK. In one embodiment of this aspect, the labeled replacement peptide comprises amino acid sequence LQSGASVVAGPR (SEQ ID NO:252) and generates a transition ion consisting of amino acid sequence SGASVVAGPR (SEQ ID NO:253) or SVVAGPR (SEQ ID NO: 254).

In another aspect of the invention, the labeled replacement peptide selectively detects or quantifies Vip3 protein and comprises the amino acid sequence of any one of SEQ ID NOS 36-73. In another aspect of the invention, the labeled replacement peptide selectively detects or quantifies Vip3 protein and generates a transition ion having an amino acid sequence selected from at least one of the peptides TCK, FEK, DVS, FTK, HK, VNI, MIV, EAK, HLK, NK, DNF, LLC, NAY, YE, SDK, NEK, DK, or VDK, or SEQ ID NO: 156-212. In one embodiment of this aspect, the labeled replacement peptide comprises amino acid sequence DGGISQFIGDK (SEQ ID NO:36) and produces a transition ion consisting of amino acid sequence SQFIGDK (SEQ ID NO:156) or amino acid sequence GDK.

In another aspect of the invention, the labeled replacement peptide selectively detects or quantifies the dmesps protein and comprises the amino acid sequence of any one of SEQ ID NOs 74-77. In another aspect of the invention, the labeled replacement peptide selectively detects or quantifies the dmesps protein and generates a transition ion having an amino acid sequence selected from at least one of SEQ ID NO 213-219 or peptide PIK. In one embodiment of this aspect, the tagged replacement peptide comprises amino acid sequence SLTAAVTAAGGNATYVLDGVPR (SEQ ID NO:257) and generates a transition ion consisting of the amino acid sequence GVPR (SEQ ID NO:258) or the amino acid sequence PR.

In another aspect of the invention, the labeled replacement peptide selectively detects or quantifies the PAT protein and comprises the amino acid sequence of any one of SEQ ID NOs: 78-86. In another aspect of the invention, the labeled replacement peptide selectively detects or quantifies the PAT protein and generates a transition ion having an amino acid sequence selected from at least one of SEQ ID NO:220-231 or the peptides DFE, DF, PER, SHR, GYK, or NFR. In one embodiment of this aspect, the labeled replacement peptide comprises amino acid sequence LGLGSTLYTHLLK (SEQ ID NO:79) and produces a transition ion consisting of the amino acid sequence YTHLLK (SEQ ID NO:220) or THLLK (SEQ ID NO: 221).

In another aspect of the invention, the labeled replacement peptide selectively detects or quantifies PMI protein and comprises the amino acid sequence of any one of SEQ ID NOS: 87-98. In another aspect of the present invention, the labeled replacement peptide selectively detects or quantifies the PMI protein and generates a transition ion having an amino acid sequence selected from the group consisting of SEQ ID NO:232-251 or at least one of the peptides LK, PVK, HN or PNK. In one embodiment of this aspect, the replacement peptide for the marker comprises amino acid sequence SALDSQQGEPWQTIR (SEQ ID NO:89) and generates a transition ion consisting of the amino acid sequence PWQTIR (SEQ ID NO:235) or GEPWQTIR (SEQ ID NO: 236).

In other aspects of the invention, the labeled replacement peptides of the invention and their resulting transition ions selectively detect or quantify the Cry1Ab protein comprising SEQ ID NO 259, or the eCry3.1Ab protein comprising SEQ ID NO 260, or the mCry3A protein comprising SEQ ID NO 261, or the Vip3 protein comprising SEQ ID NO 262, or the dmePSPS protein comprising SEQ ID NO 263, or the PAT protein comprising SEQ ID NO 264, or the PMI protein comprising SEQ ID NO 265 or SEQ ID NO 266.

In other aspects, the labeled surrogate peptide of the invention selectively detects or quantifies a target protein of the invention when the target protein is in a biological sample from a transgenic plant. In some embodiments of this aspect, the biological sample is from leaf tissue, seed, grain, pollen, or root tissue of the transgenic plant.

In other aspects of the invention, the labeled replacement peptides of the invention and their transition ions produced selectively detect or quantify Cry1Ab protein from a corn plant comprising transgenic event Bt11, or ecry3.1ab protein from a corn plant comprising transgenic event 5307, or mCry3A protein from a corn plant comprising transgenic event MIR604, or Vip3 protein from a corn plant comprising transgenic event MIR162, or cotton plant comprising transgenic event COT102, or dmesps protein from a corn plant comprising transgenic event GA21, or PAT protein from a corn plant comprising transgenic event Bt11, DAS-59122, TC1507, 411dp 4, or T25, or PMI protein from a corn plant comprising transgenic event MIR162, MIR604, 5307, or 3272.

Many different combinations of replacement peptides can be monitored and quantified simultaneously by MRM assays with one or more specific replacement peptides from Cry1Ab, ecry3.1ab, mCry3A, Vip3, dmesps, PAT, and/or PMI proteins, and thus provide a means to measure the total amount of each of those proteins in a given protein preparation obtained from a biological sample by mass spectrometry. The combination of these peptides with MRM-based assays has many applications, including quantitative peptide/protein analysis for determining expression levels at different growth stages of transgenic plants, determining expression levels in different transgenic plant tissues and organs (including but not limited to leaf tissue, seeds and kernels, pollen and root tissue), determining potential exposure levels for regulatory risk assessment, determining different levels of protein in food processing, comparative and generational studies. Broadly speaking, these seven unique surrogate peptides can be used in combination with MRM assays for a variety of applications, including agricultural applications, bioequivalence testing, biomarkers in all types of biological and non-biological matrices, diagnostic, discoverability, food, environmental, therapeutic monitoring. In some aspects of the invention, an assay cartridge is provided comprising one or more labeled surrogate peptides of the invention comprising any one of SEQ ID NOs 1-98, allowing for the simultaneous and selective detection or quantification of any one or more target proteins of the invention.

The invention also provides methods for selectively detecting or quantifying transgenic target proteins within complex biological matrices, such as biological samples from transgenic plants expressing these transgenic target proteins. Such methods comprise obtaining a sample from the transgenic plant, for example from a leaf, seed or kernel, pollen or root; extracting a protein from the plant sample; concentrating the target protein pool by reducing the amount of non-transgenic insoluble proteins in the extract; digesting soluble proteins in the extract with a selected enzyme (e.g., trypsin) to obtain an extract comprising peptide fragments, wherein the peptide fragments comprise at least one replacement peptide specific for each target transgenic protein; an assay cassette for adding SIL peptides that specifically detect a target protein, wherein each of the labeled replacement peptides has the same amino acid sequence as each of the replacement peptides of the target transgenic protein, and wherein the number of labeled replacement peptides added is equal to the number of target transgenic proteins in the mixture; concentrating the replacement peptides and labeled replacement peptides by reducing the amount of non-replacement peptides in the mixture; decomposing the peptide fragment mixture using liquid chromatography; analyzing the peptide fragment mixture using mass spectrometry, wherein detection of a transition ion fragment of a labeled surrogate peptide indicates the presence of the target transgenic protein from which the surrogate peptide was derived; and, optionally, calculating the amount of the target transgene protein in the biological sample by comparing the mass spectral signal generated by the transition ion fragment with the mass spectral signal generated by the transition ion of the labeled surrogate peptide. SIL replacement peptides derived from the transgenic proteins of the invention each have a unique transition ion in mass spectrometry-based Multiple Reaction Monitoring (MRM) assays. Thus, these peptides will produce selective MS ions due to small changes in collision energy, resulting in different degrees of ionization. For example, triple quadrupole MS can be used to generate high m/z ions with peptide specificity. As a result, the methods of the invention can provide a selectivity advantage over using lower m/z strong ionic markers known in the art, thereby reducing endogenous background.

In some aspects of the invention, the target protein that is selectively detected or quantified in the methods of the invention is a Cry1Ab protein, ecry3.1ab protein, mCry3A protein, Vip3 protein, double mutant 5-enolpyruvylshikimate-3-phosphate synthase (dmesps) protein, glufosinate acetyltransferase (PAT) protein, or phosphomannose isomerase (PMI) protein.

In other aspects of the invention, the labeled replacement peptide useful in the methods of the invention to detect or quantify the Cry1Ab protein, eCry3.1Ab protein, mCry3A protein, Vip3 protein, double mutant 5-enolpyruvylshikimate-3-phosphate synthase (dmePSPS) protein, glufosinate acetyltransferase (PAT) protein, or phosphomannose isomerase (PMI) protein comprises any one of SEQ ID NOs 1-98.

In another aspect of the methods of the present invention, the labeled replacement peptide selectively detects or quantifies Cry1Ab and comprises the amino acid sequence of any one of SEQ ID NOs 1-26. In another aspect, the labeled replacement peptide selectively detects or quantifies Cry1Ab and produces a transition ion having an amino acid sequence selected from SEQ ID NOs 99-141 or at least one of the peptides PIR, TY, VW, HR, YR, or PPR. In one embodiment of this aspect, the labeled replacement peptide comprises amino acid sequence SAEFNNIIPSSQITQIPLTK (SEQ ID NO:21) and generates a transition ion consisting of the amino acid sequence PLTK (SEQ ID NO:132) or SAEFNNII (SEQ ID NO: 133). In another aspect, Cry1Ab target protein in the biological sample is quantified by comparing the mass spectral signals generated by transition ion fragments consisting of the amino acid sequence PLTK (SEQ ID NO: 132).

In another aspect of the method of the invention, the labeled replacement peptide selectively detects or quantifies the eCry3.1Ab protein and comprises the amino acid sequence of any one of SEQ ID NOS 27-32. In another aspect of the invention, the labeled replacement peptide selectively detects or quantifies the eCry3.1Ab protein and generates a transition ion having an amino acid sequence selected from the group consisting of SEQ ID NO:142-150 or at least one of the peptides DGR, IEF or LER. In one embodiment of this aspect, the labeled replacement peptide comprises amino acid sequence TDVTDYHIDQV (SEQ ID NO:27) and generates a transition ion consisting of amino acid sequence TDYHIDQV (SEQ ID NO:142) or DYKIDQV (SEQ ID NO: 143). On the other hand, the eCry3.1Ab target protein in the biological sample was quantified by comparing the mass spectrum signals generated by the transition ion fragment consisting of the amino acid sequence TDYHIDQV (SEQ ID NO: 142). In another embodiment of this aspect, the tagged replacement peptide comprises amino acid sequence AVFNELFTSSNQIGLK (SEQ ID NO:28) and produces a transition ion consisting of amino acid sequence TSSNQIGLK (SEQ ID NO:144) or SSNQIGLK (SEQ ID NO: 145). In another aspect, the eCry3.1Ab target protein in the biological sample is quantified by comparing the mass spectral signals generated by the transition ion fragment consisting of amino acid sequence TSSNQIGLK (SEQ ID NO: 144).

In another aspect of the methods of the present invention, the labeled replacement peptide selectively detects or quantifies the protein mCry3A and comprises the amino acid sequence of any one of SEQ ID NOS 33-35. In another aspect of the method, the labeled replacement peptide selectively detects or quantifies the mCry3A protein and generates a transition ion having an amino acid sequence selected from at least one of SEQ ID NO 151-155 or the peptide IDK. In one embodiment of this aspect, the labeled replacement peptide comprises amino acid sequence LQSGASVVAGPR (SEQ ID NO:252) and generates a transition ion consisting of amino acid sequence SGASVVAGPR (SEQ ID NO:253) or SVVAGPR (SEQ ID NO: 254). In another aspect, the mCry3A target protein in the biological sample is quantified by comparing the mass spectral signals generated by the transition ion fragment consisting of amino acid sequence SGASVVAGPR (SEQ ID NO: 253).

In another aspect of the method of the invention, the labeled replacement peptide selectively detects or quantifies Vip3 protein and comprises the amino acid sequence of any one of SEQ ID NOS 36-73. In another aspect of the method, the labeled surrogate peptide selectively detects or quantifies Vip3 protein and generates a transition ion having an amino acid sequence selected from the group consisting of SEQ ID NO:156-212 or at least one of the peptides TCK, FEK, DVS, FTK, HK, VNI, MIV, EAK, HLK, NK, DNF, LLC, NAY, YE, SDK, NEK, DK, or VDK. In one embodiment of this aspect, the labeled replacement peptide comprises amino acid sequence DGGISQFIGDK (SEQ ID NO:36) and produces a transition ion consisting of amino acid sequence SQFIGDK (SEQ ID NO:156) or amino acid sequence GDK. In another aspect, the Vip3 target protein in the biological sample was quantified by comparing the mass spectra signals generated by the transition ion fragments consisting of the amino acid sequence SQFIGDK (SEQ ID NO: 156). In another embodiment of this aspect, the labeled replacement peptide comprises the amino acid sequence FTTGTDLK (SEQ ID NO:255) and generates a transition ion consisting of the amino acid sequence TGTDLK (SEQ ID NO:256) or the amino acid sequence LK. In another aspect, the Vip3 target protein in the biological sample is quantified by comparing the mass spectral signals generated by the transition ion fragments consisting of the amino acid sequence TGTDLK (SEQ ID NO: 256).

In another aspect of the methods of the invention, the labeled replacement peptide selectively detects or quantifies the dmesps protein and comprises the amino acid sequence of any one of SEQ ID NOs 74-77. In another aspect of this method, the labeled replacement peptide selectively detects or quantifies the dmesps protein and generates a transition ion having an amino acid sequence selected from at least one of SEQ ID NOs 213-219. In one embodiment of this aspect, the tagged replacement peptide comprises amino acid sequence SLTAAVTAAGGNATYVLDGVPR (SEQ ID NO:257) and generates a transition ion consisting of the amino acid sequence GVPR (SEQ ID NO:258) or the amino acid sequence PR. In another aspect, the dmesps target protein in the biological sample is quantified by comparing the mass spectral signals generated by the transition ion fragments consisting of the amino acid sequence PR.

In another aspect of the method of the invention, the labeled replacement peptide selectively detects or quantifies the PAT protein and comprises the amino acid sequence of any one of SEQ ID NOs: 78-86. In another aspect of the invention, the labeled replacement peptide selectively detects or quantifies the PAT protein and generates a transition ion having an amino acid sequence selected from at least one of SEQ ID NO:220-231 or the peptides DFE, DF, PER, SHR, GYK, or NFR. In one embodiment of this aspect, the labeled replacement peptide comprises amino acid sequence LGLGSTLYTHLLK (SEQ ID NO:79) and produces a transition ion consisting of the amino acid sequence YTHLLK (SEQ ID NO:220) or THLLK (SEQ ID NO: 221). In another aspect, the dmesps target protein in the biological sample is quantified by comparing the mass spectral signals generated by the transition ion fragments consisting of the amino acid sequence YTHLLK (SEQ ID NO: 220).

In another aspect of the method of the invention, the labeled replacement peptide selectively detects or quantifies the PMI protein and comprises the amino acid sequence of any one of SEQ ID NOS: 87-98. In another aspect of the present invention, the labeled replacement peptide selectively detects or quantifies the PMI protein and generates a transition ion having an amino acid sequence selected from the group consisting of SEQ ID NO:232-251 or at least one of the peptides LK, PVK, HN or PNK. In one embodiment of this aspect, the replacement peptide for the marker comprises amino acid sequence SALDSQQGEPWQTIR (SEQ ID NO:89) and generates a transition ion consisting of the amino acid sequence PWQTIR (SEQ ID NO:235) or GEPWQTIR (SEQ ID NO: 236).

The invention further provides a system for high throughput detection or quantification of a transgenic target protein. Such systems comprise a cassette of pre-designed labeled surrogate peptides specific for the transgenic target protein; and one or more mass spectrometers.

Various objects, features, aspects and advantages of the present invention will become more apparent from the following detailed description of preferred embodiments of the invention, along with the accompanying drawings and sequence listing.

Brief description of the sequences

1-26 are amino acid sequences of stable isotope-labeled surrogate peptides for selectively detecting and quantifying transgenic Cry1Ab protein.

27-32 are amino acid sequences of stable isotopically labeled surrogate peptides for selective detection and quantification of transgenic eCry3.1Ab proteins.

33-35 are amino acid sequences of stable isotopically-labeled surrogate peptides for the selective detection and quantification of transgenic mCry3A protein.

SEQ ID NO 36-73 is the amino acid sequence of a stable isotopically-labeled surrogate peptide for selective detection and quantification of transgenic Vip3 protein.

74-77 are amino acid sequences of stable isotopically-labeled replacement peptides for selective detection and quantification of transgenic dmesps proteins.

78-86 are amino acid sequences of stable isotope-labeled surrogate peptides for selective detection and quantification of transgenic PAT proteins.

87-98 are amino acid sequences of stable isotopically labeled surrogate peptides for selective detection and quantification of transgenic PMI proteins.

SEQ ID NOS 99-141 are amino acid sequences of transition ions of SIL-substituted peptides having SEQ ID NOS 1-26.

142-150 is the amino acid sequence of a transition product of SIL substitution peptides having SEQ ID NOS 27-32.

151-155 is the amino acid sequence of a transition product of the SIL substitution peptide having SEQ ID NO 33-35.

156-212 is the amino acid sequence of a transition product of the SIL substitution peptide having SEQ ID NOS 36-72.

213-219 is the amino acid sequence of a transition product of the SIL substitution peptide having SEQ ID NOS 74-77.

SEQ ID NO 220-231 is the amino acid sequence of a transition product of the SIL substitution peptide having SEQ ID NO 79-86.

232-251 is the amino acid sequence of a transition product of the SIL substitution peptide having SEQ ID NOS 87-98.

SEQ ID NO 252-254 is the amino acid sequence of the SIL-substitution peptide and its transition product for selective detection and quantification of the transgenic mCry3A protein.

SEQ ID NO 255-256 is the amino acid sequence of the SIL substitution peptide and the transition product for selective detection and quantification of the transgenic Vip3A protein.

SEQ ID NO 257-258 is the amino acid sequence of the SIL substitution peptide and the transition product for the selective detection and quantification of the transgenic dmePSPS protein.

259-270 is the amino acid sequence of an exemplary target transgene protein of the present invention.

Detailed Description

This description is not intended to be an exhaustive list of all the different ways in which the invention may be practiced or to add all the features in the invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Thus, the present invention contemplates that, in some embodiments of the invention, any feature or combination of features set forth herein may be excluded or omitted. Moreover, numerous variations and additions to the different embodiments suggested herein will be apparent to those skilled in the art in view of this disclosure, without departing from the present invention. Accordingly, the following description is intended to illustrate certain specific embodiments of the invention and is not intended to be exhaustive or to limit all permutations, combinations and variations thereof.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention. General references relevant to the present invention include: alwine et al (1977) Proc.Nat.Acad.Sci. [ Proc. Natl. Acad. Sci. ]74: 5350-54; baldwin (2004) mol. cell. proteomics [ molecular and cellular proteomics ]3(1) 1-9; can and Annan (1997) Overview of peptide and protein analysis by mass spectrometry [ review for peptide and protein Mass Spectrometry ]. In Current Protocols In Molecular Biology [ abstracted from the latest Association of Molecular Biology methods ], edited by Ausubel et al, New York: Weili, pp 10.21.1-10.21.27; chang et al (2000) Plant Physiol [ Plant physiology ]122(2) 295-; domon and Aebersold (2006) Science [ Science ]312(5771): 212-17; nain et al (2005) Plant mol.biol.Rep. [ Plant molecular biology guide ]23: 59-65; patterson (1998) Protein identification and characterization by mass spectrometry [ Protein identification and characterization ] In Current Protocols In Molecular Biology [ abstracted from the latest Molecular Biology methods ], edited by Ausubel et al, New York: Weili, pages 10.22.1-10.22.24; paterson and Aebersold (1995) Electrophoresis 16: 1791-1814; rajagopal and Ahern (2001) Science 294(5551) 2571-73; sesikeran and Vasanthi (2008) Asia Pac.J.Clin.Nutr.17[ J.Clonta clinical Nutrition ] supplement 1: 241-44; and Toplak et al (2004) Plant mol.biol.Rep. [ Plant molecular biology guide ]22: 237-50.

Definition of

As used herein and in the appended claims, the singular forms "a", "an" and "the" may mean one or more than one. Thus, for example, reference to "a plant" can refer to a single plant or to multiple plants.

As used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the absence of a combination when interpreted in the alternative ("or").

The term "about" is used herein to mean about, approximately, about, or around … …. When the term "about" is used in connection with a numerical range, it defines the range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to limit the numerical values to values above and below the stated values with a variation of 20%, preferably around 10% (higher or lower). With respect to temperature, the term "about" means ± 1 ℃, preferably ± 0.5 ℃. When the term "about" is used in the context of the present invention (e.g., in combination with a temperature or molecular weight value), the exact value (i.e., without "about") is preferred.

The terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

As used herein, the transitional phrase "consisting essentially of … …" (and grammatical variants) means that the scope of the claims is to be read as encompassing the specified materials or steps recited in the claims as well as those that do not materially alter one or more of the basic and novel features of the claimed invention. Thus, the term "consisting essentially of … …" when used in the claims of this invention is not intended to be construed as equivalent to "comprising".

As used herein, the term "Cry protein" refers to an insecticidal protein in the form of a globular protein molecule that accumulates as a protoxin in crystalline form under natural conditions during the sporulation phase of the growth cycle of a bacillus species (e.g., bacillus thuringiensis). The terms "Cry toxin" and "delta-endotoxin" can be used interchangeably with the term "Cry protein". The current nomenclature for the Cry proteins and the genes encoding them is based on amino acid sequence homology (Crickmore et al (1998), Microbiol. mol. biol. Rev. [ review of microbial molecular biology ],62: 807-. In the art-recognized classification, each Cry protein is assigned a unique name that incorporates a primary level (arabic numeral), a secondary level (capital letters), a tertiary level (lowercase letters), and a quaternary level (another arabic numeral). For example, according to Crickmoe et al, two Cry proteins having < 45% homology will be assigned unique primary ranks, such as Cry1 and Cry 2. Two Cry proteins with > 45% but < 70% homology will accept the same primary grade, but will be assigned different secondary grades, e.g., Cry1A and Cry 1B. Two Cry proteins with 70% to 95% homology will be assigned the same primary and secondary ranks, but will be assigned different tertiary ranks, such as Cry1Aa and Cry1 Ab. And two Cry proteins with > 95% but < 100% homology will be assigned the same primary, secondary and tertiary grades, but will be assigned different quaternary grades, e.g., Cry1Ab1 and Cry1Ab 2.

As used herein, "Cry 1Ab protein" means an insecticidal crystal protein derived from bacillus thuringiensis, whether naturally occurring or synthetic, comprising an amino acid sequence at least 96% identical to the holotype Cry1Ab amino acid sequence (according to Crickmore et al (supra)) and disclosed on the internet site "life si. Examples of Cry1 proteins (having accession numbers) include, but are not limited to, Cry1Ab (AAA22330), Cry1Ab (AAA22613), Cry1Ab (AAA22561), Cry1Ab (BAA00071), Cry1Ab (CAA28405), Cry1Ab (AAA22420), Cry1Ab (CAA31620), Cry1Ab (AAA22551), Cry1Ab (CAA38701), Cry1Ab (a29125), Cry1Ab (I12419), Cry1Ab (AAC64003), Cry1Ab (AAN76494), Cry1Ab (AAG 168jn77), Cry1Ab (AAO13302), Cry1Ab (AAK55546), Cry1Ab (AAT46415), Cry1Ab (AAQ88259), Cry1Ab (AAW 31), Cry1Ab (ABB72460), Cry1Ab KT 18384), Cry1Ab 668 (ABW87320), Cry 1(Cry 4377), Cry1Ab (HQ 43254), Cry1Ab (HQ 43259), Cry1Ab (HQ 31), Cry1Ab (Cry 135253), Cry1Ab 1351 Ab (Cry) and Cry 7785), Cry1Ab (Cry 1357785), Cry1Ab (Cry 7785), Cry 77250). An illustrative example of a Cry1Ab protein of the invention is represented by SEQ ID NO 259.

As used herein, the term "Cry 3" refers to an insecticidal protein that shares a high degree of sequence identity or similarity with a previously described sequence classified as Cry3 (according to Crickmore et al (supra)), examples of which are disclosed on the internet website "life scissi. susex. ac. uk/home/Neil _ Crickmore/Bt/" and include (with accession numbers) Cry3Aa1(AAA22336), Cry3Aa2(AAA22541), Cry3Aa3(Caa68482), Cry3Aa4(AAA22542), Cry3Aa5(AAA50255), Cry3Aa6(AAC43266), Cry3Aa7(CAB41411), Cry3Aa8(AAS79487), Cry3Aa9(AAW 659), Cry3Aa10 (aa29411), Cry3Aa11 (caw 41327), Cry3Aa 495963 (aca) and Cry 3538 (Cry 9827 b), Cry3a 364676, Cry3 a). Cry3 proteins that have been engineered by insertion, substitution, or deletion of amino acids are referred to herein as "modified Cry3 proteins" or "mCry 3 proteins". Such "modified Cry3 proteins" typically have enhanced activity against certain insect pests, such as corn rootworm (diabrotica spp.) as compared to the wild-type Cry3 protein from which the "modified Cry3 protein" is derived. An example of a "modified Cry3 protein" is "mCry 3A" represented by the amino acid sequence of SEQ ID NO: 262. Other examples of "modified Cry 3" proteins include, but are not limited to, the "mCry 3A protein" disclosed in U.S. patent 8,247,369, the "mCry 3A protein" disclosed in U.S. patent 9,109,231, and the "mCry 3B protein" disclosed in U.S. patent 6,060,594.

The term "ecry 3.1ab" refers to an engineered hybrid insecticidal protein comprising in the N-terminal to C-terminal direction the N-terminal region of Cry3A fused to the C-terminal region of a Cry1Aa or Cry1Ab protein as described in U.S. patent 8,309,516. An example of "eCry 3.1Ab protein" is represented by the amino acid sequence of SEQ ID NO: 260.

As used herein, the term transgenic "event" refers to a recombinant plant produced by transforming and regenerating a single plant cell with heterologous DNA (e.g., an expression cassette comprising a gene of interest). The term "event" refers to the original transformant and/or progeny of the transformant that comprise the heterologous DNA. The term "event" also refers to progeny resulting from a sexual outcross (sexual outcross) between the transformant and another maize line. Even after repeated backcrossing to a recurrent parent, the insert DNA and flanking DNA from the transformed parent are present at the same chromosomal location in the progeny of the cross. Typically, transformation of plant tissue results in multiple events, each of which represents the insertion of a DNA construct into a different location in the genome of a plant cell. The particular event is selected based on the expression of the transgene or other desired characteristic. Non-limiting examples of such transgenic events of the invention include "event Bt 11", which comprises cry1Ab and pat genes, and is described in US 6114608 (also referred to as "Bt 11 event" or "Bt 11 only"); "event 5307" comprising ecry3.1ab and PMI genes and described in US 8466346 (also referred to as "5307 event" or simply "5307"); "event MIR 604", which comprises mCry3A and a PMI gene, and is described in US 7361813 (also referred to as "MIR 604 event" or simply "MIR 604"); "event MIR 162", which comprises Vip3A and the PMI gene, and is described in US 8232456 (also referred to as "event MIR 162" or simply "MIR 162"); "event GA 21" comprising the dmesps gene, and described in US 6566587 (also referred to as "GA 21 event" or simply "GA 21"); "event 3272", which comprises alpha-amylase 797E and the PMI gene, and is described in US 7635799 (also referred to as "3272 event" or simply "3272"); an "event MON 810" comprising Cry1Ab and described in US 6713259 (also referred to as "MON 810 event" or simply "MON 810"); "event MON 89034" comprising cry1a.105 and Cry2Ab genes and described in US 8062840 (also referred to as "MON 89034 event" or simply "MON 89034"); "event TC 1507" comprising Cry1F and the PAT gene, and described in US 7288643 (also referred to as "TC 1507 event" or simply "TC 1507"); an "event DAS 59122" comprising Cry34/Cry35 and PAT genes, and described in US 7323556 (also referred to as "DAS 59122 event" or simply "DAS 59122") and "event DP 4114" comprising Cry1F, Cry34/Cry35 and PAT genes, and described in US 9790561 (also referred to as "DP 4114 event" or simply "DP 4114").

As used herein, the term "hybrid Cry protein" is an engineered insecticidal protein that does not occur in nature, and at least a portion thereof comprises at least one 27% contiguous Cry1Ab protein amino acid sequence. The 27% limit is calculated by dividing the number of consecutive Cry1Ab amino acids in the hybrid Cry protein by the total number of amino acids in the hybrid Cry protein. For example, the hybrid Cry protein eCry3.1Ab (SEQ ID NO:261) has 174 Cry1Ab amino acids (position 480. cndot. 653) and a total of 653 amino acids. Thus, ecry3a.1ab has at least one 27% contiguous amino acid sequence of Cry1Ab protein. Another example of a hybrid Cry protein Cry1A.105 according to the invention is represented by SEQ ID NO 267.

"dmesps" (5-enolpyruvylshikimate-3-phosphate synthase) is an engineered protein that confers tolerance to glyphosate herbicides on plants, as described in PCT publication No. WO 97/04103. An illustrative example of a dmesps of the present invention is represented by SEQ ID NO: 263.

As used herein, "highly related insecticidal proteins" refers to proteins having at least 95% overall sequence identity, or proteins having a common motif with at least 80% sequence identity. Examples of "highly related" insecticidal proteins include Cry1Ab (SEQ ID NO:259) and eCry3.1Ab (SEQ ID NO:260) which have a common motif with at least 80% sequence identity, and eCry3.1Ab (SEQ ID NO:260) and mCry3A (SEQ ID NO:261) which have a common motif with at least 80% sequence identity.

The term "isolated" nucleic acid molecule, polynucleotide or toxin is a nucleic acid molecule, polynucleotide or toxic protein that is no longer present in its natural environment. The isolated nucleic acid molecule, polynucleotide or toxin of the invention may be present in purified form or may be present in a recombinant host, such as a transgenic bacterial cell or a transgenic plant.

As used herein, the general term "mass spectrometry" refers to any suitable mass spectrometry method, apparatus or configuration, including, for example, electrospray ionization (ESI), matrix assisted laser desorption/ionization (MALDI) MS, MALDI-time of flight (TOF) MS, Atmospheric Pressure (AP) MALDI MS, vacuum MALDI MS, tandem MS, or any combination thereof. Mass spectrometry devices measure the molecular weight of a molecule (as a function of its mass-to-charge ratio) by measuring the flight path of the molecule in a set of magnetic and electric fields. The mass-to-charge ratio is a physical quantity widely used in the electrodynamics of charged particles. The mass to charge ratio of a particular peptide can be calculated in advance by one skilled in the art. Two particles with different mass-to-charge ratios do not move along the same path in a vacuum when subjected to the same electric and magnetic fields. The invention specifically includes the use of High Performance Liquid Chromatography (HPLC) followed by tandem MS analysis of the peptide. In "tandem mass spectrometry," a surrogate peptide can be filtered in an MS instrument and then fragmented to produce one or more "transition ions" which are analyzed (detected and/or quantified) in a second MS procedure.

A detailed overview of mass spectrometry methods and apparatus can be found in the following references, which are incorporated herein by reference: can and Annan (1997) Overview of peptide and protein analysis by mass spectrometry [ review for peptide and protein Mass Spectrometry ]. In Current Protocols In Molecular Biology [ abstracted from the latest Association of Molecular Biology methods ], edited by Ausubel et al, New York: Weili, pp 10.21.1-10.21.27; paterson and Aebersold (1995) Electrophoresis 16: 1791-1814; patterson (1998) Protein identification and characterization by mass spectrometry [ Protein identification and characterization ] In Current Protocols In Molecular Biology [ abstracted from the latest Molecular Biology methods ], edited by Ausubel et al, New York: Weili, pages 10.22.1-10.22.24; and Domon and Aebersold (2006) Science 312(5771): 212-17.

Peptides are short polymers made up of alpha-amino acids linked in a defined order. Peptides may also be produced by digestion of polypeptides (e.g., proteins) with proteases.

A "plant" is any plant, particularly a seed plant, at any stage of development.

A "plant cell" is the structural and physiological unit of a plant, comprising protoplasts and a cell wall. The plant cells may be in the form of isolated individual cells or cultured cells, or as part of a higher order tissue unit, such as, for example, a plant tissue, plant organ, or whole plant.

By "plant cell culture" is meant a culture of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissue, pollen tubes, ovules, embryo sacs, zygotes, and embryos at different developmental stages.

"plant material" means leaves, stems, roots, flowers or parts of flowers, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.

A "plant organ" is a distinct and distinct, structured and differentiated part of a plant, such as a root, stem, leaf, bud, or embryo.

"plant tissue" as used herein means a group of plant cells organized into structural and functional units. Including any plant tissue in a plant or in culture. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue cultures, and any group of plant cells organized into structural and/or functional units. The use of this term in combination or alone with any particular type of plant tissue as listed above or otherwise encompassed by this definition is not intended to exclude any other type of plant tissue.

As used herein, the term "surrogate peptide" refers to a peptide derived from a target transgenic protein by proteolytic digestion, e.g., trypsin digestion, the target transgenic protein acting in a mass spectrometry assay to produce one or more transition ions that, when the target transgenic protein is accompanied by the presence of one or more other transgenic proteins and/or non-transgenic proteins in a complex biological matrix (such as a sample from a transgenic plant), differentially detects and/or quantifies the target transgenic protein in combination with the surrogate peptide, and does not detect and/or quantify the one or more other transgenic proteins or the non-transgenic proteins in the biological matrix. The "substitute peptide" may also be referred to as a "signature peptide" of the target transgenic protein. For example, a Cry1Ab replacement peptide of the invention produces one or more transition ions that differentially detect and/or quantify the target Cry1Ab transgenic insecticidal protein in a composite biological matrix in combination with a Cry1 Ab-replacement peptide when the Cry1Ab transgenic protein is accompanied by the presence of one or more non-Cry 1Ab transgenic proteins, such as an ecry3.1ab insecticidal protein of the invention or an mCry3A insecticidal protein and/or a non-transgenic protein. In another example, an ecry3.1ab replacement peptide of the invention in combination with an ecry 3.1ab-replacement peptide produces one or more transition ions that differentially detect and/or quantify a target ecry3.1ab transgene protein in a composite biological matrix when the ecry3.1ab transgene protein is accompanied by the presence of one or more non-ecry 3.1ab transgene proteins, such as Cry1Ab or mCry3A of the invention and/or non-transgene proteins in the composite biological matrix. According to embodiments of the invention, two or more labeled surrogate peptides of the invention can be used simultaneously in a mass spectrometry assay to detect and/or quantify two or more target transgenic proteins in a complex biological matrix.

A "labeled surrogate peptide" is a non-naturally occurring surrogate peptide that is labeled to facilitate detection of the surrogate peptide in a mass spectrometry assay. For example, the label may be a stable isotopically labeled amino acid (SIL), such as lysine, isoleucine, valine, or arginine. Thus, a SIL-labeled replacement peptide has the same amino acid sequence as an unlabeled replacement peptide, except that one or more amino acids of the replacement peptide are heavily isotopically labeled. For example, the replacement peptide SAEFNNIIPSSQITQIPLTK (SEQ ID NO:21) was labeled with heavy lysine (K) and may be designated SAEFNNIIPSSQITQIPLTK [ C13N15-K ]; this replacement peptide TDVTDYHIDQV (SEQ ID NO:27) was labeled with heavy valine (V) and may be designated TDVTDYHIDQV [ C13N15-V ]; this replacement peptide LQSGASVVAGPR (SEQ ID NO:252) was labeled with arginine (R) and may be named LQSGASVVAGPR [ C13N15-R ]; this replacement peptide DGGISQFIGDK (SEQ ID NO:36) was labeled with heavy lysine (K) and may be named DGGISQFIGDK [ C13N15-K ]; the replacement peptide FTTGTDLK (SEQ ID NO:255) is labeled with lysine (K) and may be named FTTGTDLK [ C13N15-K ]; this replacement peptide SLTAAVTAAGGNATYVLDDGVPR (SEQ ID NO:257) was labeled with heavy arginine (R) and may be named SLTAAVTAAGGNATYVLDDGVPR [ C13N15-R ]; this replacement peptide LGLGSTLYTHLLK (SEQ ID NO:79) was labeled with heavy lysine and may be named LGLGSTLYTHLLK [ C13N15-K ]; this replacement peptide SALDSQQGEPWQTIR (SEQ ID NO:89) was labeled with heavy arginine (R) and may be named SALDSQQGEPWQTIR [ C13N15-R ], and so on.

As described in PCT publication No. WO 87/05629, the "PAT" (glufosinate N-acetyltransferase) protein confers tolerance to glufosinate herbicides on plants. An illustrative example of the PAT protein of the present invention is represented by SEQ ID NO 264.

The "PMI" (mannose 6-phosphate isomerase) protein confers on plant cells the ability to utilize mannose, as described in US 5767378. Illustrative examples of PMI proteins of the present invention are represented by SEQ ID NO:265 and SEQ ID NO: 266.

As used herein, the term "stacked" refers to the presence of multiple heterologous polynucleotides or transgenic proteins or transgenic events incorporated in the genome of a plant.

As used herein, "target protein" refers to a protein, typically a transgenic protein, intended for selective detection and/or quantification by a labeled surrogate peptide when the target protein is in a complex biological matrix.

As used herein, the term "transgenic protein" means a protein or peptide produced in a non-native form, location, organism, or the like. Thus, a "transgenic protein" may be a protein having the same amino acid sequence as a naturally occurring protein, or it may be a protein having a non-naturally occurring amino acid sequence. For example, a Cry1Ab protein having the same amino acid sequence as a wild-type Cry1Ab protein from bacillus thuringiensis (an organism that produces native Cry 1Ab) is a "transgenic protein" when produced in a transgenic plant or bacterium.

Nucleotides are referred to herein by the following standard abbreviations: adenine (a), cytosine (C), thymine (T), and guanine (G). Amino acids are also represented by the following standard abbreviations: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gln; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (Ile; 1), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

The present invention includes compositions, methods, and systems useful for performing mass spectrometry for differentially detecting and/or quantifying one or more target transgenic proteins in a mixture comprising transgenic and non-transgenic proteins in a complex biological sample derived from a transgenic plant, e.g., a biological sample from leaves, stems, roots, pollen, and seeds of one or more transgenic plants, each biological sample possibly having a different effect on the mass spectrometry results. The compositions, methods and systems of the invention may also be used to test non-transgenic plants that are at risk of contamination with transgenes from neighboring plants, for example by cross-pollination. By these embodiments, the incidental presence of a transgene can be monitored and limited. In other embodiments, the methods disclosed herein can be used to screen the results of plant transformation procedures to identify transformants that exhibit the desired expression characteristics of the transgenic protein.

The skilled artisan can select the preference for the particular target protein to be analyzed at his or her discretion. Such proteins may be, but are not limited to, those from plants, animals, bacteria, yeast, etc., and may be proteins found in non-transformed cells or in transformed cells. Particularly suitable proteins for expression in transgenic plants are those that confer tolerance to herbicides, insects, or viruses, as well as genes that provide improved nutritional value, increased yield, drought tolerance, nitrogen utilization, production of useful industrial compounds, processing of plant characteristics, or potential for bioremediation. Examples of such proteins include insecticidal crystal proteins (i.e. Cry proteins) and vegetative insecticidal proteins (i.e. Vip from bacillus thuringiensis) or engineered proteins derived therefrom (for conferring insect resistance), herbicide tolerance proteins (such as5 '-enolpyruvate-3' -phosphoshikimate synthase (EPSPS) or glufosinate acetyltransferase (PAT) or selectable marker proteins such as phosphomannose isomerase (PMI) — as will be readily understood by a person skilled in the art, any protein conferring a desired trait may be expressed in a plant cell using recombinant DNA technology and may therefore be a target transgenic protein according to the present invention.

More specifically, the present invention provides compositions, diagnostic methods and systems for performing the diagnostic methods that allow for specific differential detection and/or quantification of Cry1Ab, ecry3.1ab, mCry3A, Vip3, dmesps, PAT and PMI transgenic proteins in complex biological matrices in samples from transgenic plant tissues (such as leaves, roots, stems, pollen, seeds or grains). The compositions, diagnostic methods and systems of the present invention are particularly useful for differentially detecting and/or quantifying highly similar transgenic insecticidal proteins, such as Cry1Ab, mCry3A and ecry3.1ab, in complex biological samples comprising the transgenic insecticidal proteins. The current state of the art is that commercially available antibody-based immunoassays cannot be used to differentially detect Cry1Ab proteins from hybrid Cry proteins engineered using the amino acid sequences of a large number of Cry1Ab proteins (when the two proteins are in the same biological sample) because of the high cross-reactivity of antibodies between these two types of proteins. For example, antibodies raised against the wild-type Cry1Ab used in a Cry1Ab detection immunoassay are cross-reactive with a hybrid Cry protein having as few as 27% of its amino acids derived from the wild-type Cry1Ab protein (when both proteins are in the same biological sample). Thus, for example, quantification of wild-type Cry1Ab in such a composite biological sample may be confounded by the presence of one or more non-target wild-type Cry proteins or non-target hybrid Cry proteins. Furthermore, due to the cross-reactivity of antibodies to Cry1Ab protein and hybrid Cry proteins in transgenic plant products, detection using expressed proteins is difficult for identity preservation of commercial transgenic plant products comprising wild-type Cry1Ab and one or more of the subject hybrid Cry proteins. The methods and compositions disclosed herein provide solutions to these problems and rely on surrogate peptides from the target transgenic protein and transition ions derived from the surrogate peptides for differential detection and/or quantification of the target protein, even when the target protein is in a mixture of other very closely related transgenic and non-transgenic proteins.

The accuracy of target protein quantification by mass spectrometry multiple reaction monitoring assays (MRM) is completely dependent on the selection of the appropriate surrogate peptide and the ability of the surrogate peptide/transition ion combination to discriminate against the target protein. Many different combinations of the replacement peptides of the invention can be monitored and quantified simultaneously by MRM assays with one or more specific peptides from Cry1Ab, ecry3.1ab, mCry3A, Vip3, dmesps, PAT and/or PMI proteins, and thus provide a means to identify and quantify each target protein in a given biological sample by mass spectrometry. The replacement peptides for the seven target proteins may constitute a cassette to quantify each respective target protein, i.e., Cry1Ab, ecry3.1ab, mCry3A, Vip3, dmesps, PAT, and/or PMI. The available surrogate peptides that make up the cassette can be analyzed in a single MRM assay, alone or in any combination, or in multiple MRM assays.

The surrogate peptide binding MRM-based assays of the invention have numerous applications, including quantitative peptide/protein analysis for determining expression levels at different growth stages, determining potential exposure levels for environmental risk assessment, determining different levels of target proteins in food processing, determining expression levels in comparative studies, and comparing expression levels in generational studies. Broadly speaking, these seven protein unique surrogate peptides can be used in combination with MRM assays to monitor or quantify selectable markers, herbicide tolerance, or insecticidal status in a single transgenic event or in a breeding stack of multiple transgenic events that may be in a particular tissue (i.e., leaf, root, grain, pollen).

The MRM-based assay can quantify or measure the relative or absolute levels of particular replacement peptides from proteins including Cry1Ab, ecry3.1ab, mCry3A, Vip3, dmesps, PAT, and/or PMI. The relative quantitative levels of these proteins can be determined by MRM measurements by comparing the characteristic peak areas to each other. The relative levels of individual Cry1Ab, ecry3.1ab, mCry3A, Vip3, dmesps, PAT, and/or PMI replacement peptides can be quantified from different samples or tissue types. In general, relative quantitative levels are determined by comparing peptide abundance in MRM measurements with Stable Isotopically Labeled (SIL) synthetic peptide analogs as internal standards for each target surrogate peptide. In contrast to what is typically taught in the art, typically by incorporation¹³C₆ ¹⁵N₂]Lysine or [ alpha ], [ alpha ]¹³C₆ ¹⁵N₄]Arginine serves to label the SIL peptide, but may also include other amino acids, such as isoleucine and valine. The SIL standard is required to have high purity and should be quantitatively standardized by amino acid analysis. In contrast to what is typically taught in the art, the SILs of the present invention are added to the sample immediately after protein digestion and thus used to correct for subsequent analysis steps. The SIL co-elutes with the unlabeled surrogate peptide in liquid chromatography separation and shows the same MS/MS fragmentation pattern, but differs only in mass due to the isotopic labeling. The mass shift results of the labeled surrogate peptide and the product ion allow the mass spectrometer to distinguish between unlabeled and labeled peptides. Since complex peptide digests typically contain multiple sets of co-eluting transitions that may be mistaken for the target peptide, co-elution of the isotopically labeled standards identifies the correct signal and provides optimal protection against false positive quantitation. Since a known concentration of the spiked SIL standard was spiked into each sample, the relative amounts of each corresponding replacement peptide from different target proteins of Cry1Ab, ecry3.1ab, mCry3A, Vip3, dmesps, PAT, and/or PMI could be determined. Since one or more individual peptides can be performed relative to the amount of another peptide or peptides within a sample or between samplesRelative quantification, therefore, the relative amount of peptides present can be determined by determining whether the peak areas are relative to each other within the biological sample. The relative quantitative data between different samples derived from the individual characteristic peak areas is typically normalized to the amount of protein analyzed for each sample. Relative quantification of multiple peptides from multiple proteins can be performed simultaneously in one sample and/or multiple samples to further understand the relative protein amounts of one peptide/protein relative to other peptides/proteins.

Absolute quantitative levels of Cry1Ab, ecry3.1ab, mCry3A, Vip3, mEPSPS, PAT, and/or PMI can be determined by MRM-based assays by comparing the characteristic peak areas of individual surrogate peptides from the corresponding proteins in a biological sample to known amounts of one or more internal standards in the sample. This can be achieved by adding known concentrations of these proteins to a negative control matrix that does not contain the target protein. The Multiple Reaction Monitoring (MRM) assay comprises weighing the non-transgenic sample with the exact additive standard concentration of each of the seven proteins; extracting and homogenizing the sample in lysis buffer; centrifuging the sample to separate soluble and insoluble proteins to enrich and reduce the complexity of the extraction; digesting a soluble protein sample with trypsin (the tissue or biological sample may be treated with one or more proteases including but not limited to trypsin, chymotrypsin, pepsin, endoproteases Asp-N and Lys-C for a period of time sufficient to digest the sample), centrifuging the sample, adding a fixed concentration of SIL peptide (in absolute quantification, the SIL serves as an indicator); desalting by solid phase extraction with cation exchange to minimize matrix effects or interference and reduce ion suppression; and the sample was analyzed by liquid chromatography in combination with tandem mass spectrometry. Typically, a mass spectrometer, typically an ion trap mass spectrometer or another form of mass spectrometer capable of performing global analysis, is performed to identify as many peptides as possible from a single composite protein/peptide lysate for analysis. While MRM-based assays can be developed and performed on any type of mass spectrometer, it is generally believed that the most advantageous instrument platform for MRM assays is the triple quadrupole instrument platform. The replacement peptides and SIL of interest unique to these seven proteins were measured by LC-MS/MS. The peak area ratio (peak area of the replacement peptide/peak area of the corresponding SIL peptide) was determined for each peptide of interest. The peak area ratios were used to back-calculate the concentrations of the seven proteins of interest from the calibration curve. Absolute quantification of many peptides is possible, allowing for the quantitative determination of multiple proteins in a single sample and/or in multiple samples simultaneously, giving insight into the absolute amount of protein in a single biological sample or large sample set.

In some embodiments, the invention includes a labeled surrogate peptide that functions in a mass spectrometry assay, such as a multiple reaction monitoring assay, to selectively detect or quantify a target transgenic protein selected from the group consisting of: a Cry1Ab protein, an ecry3.1ab protein, a mCry3A protein, a Vip3 protein, a double mutant 5-enolpyruvylshikimate-3-phosphate synthase (dmesps) protein, a glufosinate acetyltransferase (PAT) protein, and a phosphomannose isomerase (PMI) protein in a mixture of transgenic and non-transgenic proteins in one or more biological samples from one or more transgenic plants, the replacement peptide comprising a marker and an amino acid sequence selected from the group consisting of: GSAQGIEGSIR (SEQ ID NO:1), IVAQLGQGVYR (SEQ ID NO:2), TLSSTLYR (SEQ ID NO:3), DVSVFGQR (SEQ ID NO:4), TYPR (SEQ ID NO:5), TVSQLTR (SEQ ID NO:6), WYNTGTGLER (SEQ ID NO:7), EWEADPTNPALR (SEQ ID NO:8), VWPGPDSR (SEQ ID NO:9), APMFSWIHR (SEQ ID NO:10), WGFDAATINSR (SEQ ID NO:11), NQASSR (SEQ ID NO:12), IEEFAR (SEQ ID NO:13), SGFSNSSVSIIR (SEQ ID NO:14), LSHVSMFR (SEQ ID NO:15), 23 73 (SEQ ID NO:16), LEGLSNLYQIYAESFR (SEQ ID NO:17), YNFR (SEQ ID NO:18), YNDTRR (SEQ ID NO:19), SPHLMDILNSITIYTDAHR (SEQ ID NO:20), SAEFNNIIPSSQITQIPLTK (SEQ ID NO:21), QGFR (SEQ ID NO:22), SEQ ID NO: MDNNPNINECIPYNCLSNPEVEVLGGER (SEQ ID NO:24), SEQ ID NO:24), RPFNIGINNQQLSVLDGTEFAYGTSSNLPSAVYR (SEQ ID NO:25), SGTVDSLDEIPPQNNNVPPR (SEQ ID NO:26), TDVTDYHIDQV (SEQ ID NO:27), AVNELFTSSNQIGLK (SEQ ID NO:28), ITQLPLTK (SEQ ID NO:29), GLDSTTK (SEQ ID NO:30), QCAGIRPYDGR (SEQ ID NO:31), IEFVPAEVTFEAEYDLER (SEQ ID NO:32), ITQLPLVK (SEQ ID NO:33), MTADNNTEALDSSTTK (SEQ ID NO:34), VYIDK (SEQ ID N: 31)O35), DGGISQFIGDK (SEQ ID NO:36), LITTLTCK (SEQ ID NO:37), ELLLATDLSNK (SEQ ID NO:38), FEELTFATETSSK (SEQ ID NO:39), EVLFEK (SEQ ID NO:40), TASELITK (SEQ ID NO:41), DVSEMFTTK (SEQ ID NO:42), LLGLADIDYTSIMNEHLNK (SEQ ID NO:43), IDFTK (SEQ ID NO:44), TDTGGDLTLDEILK (SEQ ID NO:45), DIMNMIFK (SEQ ID NO:46), ALYVHK (SEQ ID NO:47), VNILPTLSNTFSNPNYAK (SEQ ID NO:48), ITSMLSDVIK (SEQ ID NO:49), QNLQLDSFSTYR (SEQ ID NO:50), DSLSEVIYGDMDK (SEQ ID NO:51), MIVEAKPGHALIGFEISNDSITVLK (SEQ ID NO:52), VYFSVSGDANVR (SEQ ID NO:53), NQQLLNDISGK (SEQ ID NO:54), VESSEAEYR (SEQ ID NO:55), SGAK (SEQ ID NO:56), DGSPADILDELTELTELAK (SEQ ID NO: 6357), VYE NO:58 (SEQ ID NO:58), and VYE NO:58), LDAINTMLR (SEQ ID NO:59), GKPSIHLK (SEQ ID NO:60), DENTGYIHYEDTNNNLEDYQTINK (SEQ ID NO:61), DNFYIELSQGNNLYGGPIVHFYDVSIK (SEQ ID NO:62), LLCPDQSEQIYYTNNIVFPNEYVITK (SEQ ID NO:63), SQNGDEAWGDNFIILEISPSEK (SEQ ID NO:64), NAYVDHTGGVNGTK (SEQ ID NO:65), LDGVNGSLNDLIAQGNLNTELSK (SEQ ID NO:66), IANEQNQVLNDVNNK (SEQ ID NO:67), YEVTANFYDSSTGEIDLNK (SEQ ID NO:68), QNYALSLQIEYLSK (SEQ ID NO:69), QLQEIISDK (SEQ ID NO:70), LLSPELINTNNWTSTGSTNISGNTLTLYQGGR (SEQ ID NO:71), YVNEK (SEQ ID NO:72), QNYQVDK (SEQ ID NO:73), MAGAEEIVLQPIK (SEQ ID NO:74), FPVEDAK (SEQ ID NO:75), SGTVK (SEQ ID NO:76), ILLLAALSEGTTVVDNLLNSEDVHYMLGALR (SEQ ID NO:77), DFELPAPPRPVRPVTQI (SEQ ID NO:78), LGLGSTLYTHLLK (SEQ ID NO:79), SEQ ID NO: 3680), PER (SEQ ID NO: HGGWHDVGFWQR), PER NO: 3982 (SEQ ID NO: 3982), and/18, TEPQTPQEWIDDLER (SEQ ID NO:83), AAGYK (SEQ ID NO:84), YPWLVAEVEGVVAGIAYAGPWK (SEQ ID NO:85), RPVEIRPATAADMAAVCDIVNHYIETSTVNFR (SEQ ID NO:86), ENAAGIPMDAAER (SEQ ID NO:87), ALAILK (SEQ ID NO:88), SALDSQQGEPWQTIR (SEQ ID NO:89), GSQQLQLKPGESAFIAANESPVTVK (SEQ ID NO:90), FEAKPANQLLTQPVK (SEQ ID NO:91), STLLGEAVAK (SEQ ID NO:92), LINSVQNYAWGSK (SEQ ID NO:93), HNSEIGFAK (SEQ ID NO:94), VLCAAQPLSIQVHPNK (SEQ ID NO:95), TALTELYGMENPSSQPMAELWMGAHPK (SEQ ID NO:96), LSELFASLLNMQGEEK (SEQ ID NO:97) and QGAELDFPIPVDDFAFSLHDLSDK (SEQ ID NO: 98). In other embodiments, the peptide is produced by incorporating a stable isotopically-labeled (SIL) amino acidTo label the replacement peptide. In other embodiments, by incorporation¹³C₆ ¹⁵N₂]Lysine, [ 2 ]¹³C₆ ¹⁵N₂]Isoleucine, [ 2 ]¹³C₆ ¹⁵N₂]Valine or [ alpha ], [ alpha ]¹³C₆ ¹⁵N₂]Arginine to label the SIL peptide.

In some embodiments, the labeled replacement peptide selectively detects or quantifies a Cry1Ab protein in a mixture of transgenic and non-transgenic proteins and comprises an amino acid sequence selected from the group consisting of: GSAQGIEGSIR (SEQ ID NO:1), IVAQLGQGVYR (SEQ ID NO:2), TLSSTLYR (SEQ ID NO:3), DVSVFGQR (SEQ ID NO:4), TYPR (SEQ ID NO:5), TVSQLTR (SEQ ID NO:6), WYNTGTGLER (SEQ ID NO:7), EWEADPTNPALR (SEQ ID NO:8), VWPGPDSR (SEQ ID NO:9), APMFSWIHR (SEQ ID NO:10), WGFDAATINSR (SEQ ID NO:11), NQASSR (SEQ ID NO:12), IEEFAR (SEQ ID NO:13), SGFSNSSVSIIR (SEQ ID NO:14), LSHVSMFR (SEQ ID NO:15), 23 73 (SEQ ID NO:16), LEGLSNLYQIYAESFR (SEQ ID NO:17), YNFR (SEQ ID NO:18), YNDTRR (SEQ ID NO:19), SPHLMDILNSITIYTDAHR (SEQ ID NO:20), SAEFNNIIPSSQITQIPLTK (SEQ ID NO:21), QGFR (SEQ ID NO:22), SEQ ID NO: MDNNPNINECIPYNCLSNPEVEVLGGER (SEQ ID NO:24), SEQ ID NO:24), RPFNIGINNQQLSVLDGTEFAYGTSSNLPSAVYR (SEQ ID NO:25) and SGTVDSLDEIPPQNNNVPPR (SEQ ID NO: 26).

In other embodiments, Cry1 Ab-specifically labeled alternative peptides of the invention generate transition ions having an amino acid sequence selected from the group consisting of: GIEGSIR (SEQ ID NO:99), EGSIR (SEQ ID NO:100), AQLGQGVYR (SEQ ID NO:101), GQGVGR (SEQ ID NO: 102); SSLYR (SEQ ID NO:103), STLYR (SEQ ID NO:104), SVFGQR (SEQ ID NO:105), FGQR (SEQ ID NO:106), PIR, TY, SQLTR (SEQ ID NO:107), QLTR (SEQ ID NO:108), NTGLER (SEQ ID NO:109), YNTTGLER (SEQ ID NO:110), PTNPALR (SEQ ID NO:111), DPTNPALR (SEQ ID NO:112), GPDSR (SEQ ID NO:113), VW, HR, SWIHR (SEQ ID NO:114), ATINSR (SEQ ID NO:115), DAATINSR (SEQ ID NO:116), AISR (SEQ ID NO:117), ISR, EFAR (SEQ ID NO:118), EEFAR (SEQ ID NO:119), SNSSVSIIR (SEQ ID NO:120), SSVSIIR (SEQ ID NO:121), SMFR (SEQ ID NO:122), VSMFR (SEQ ID NO:123), GSFR (SEQ ID NO:125), SEQ ID NO:124, FDFR (SEQ ID NO:124), and so, YAESFR (SEQ ID NO:126), LEG, NQFR (SEQ ID NO:127), QFR, DLTR (SEQ ID NO:128), NDLTR (SEQ ID NO:129), TIYTDAHR (SEQ ID NO:130), YTDAHR (SEQ ID NO:131), PLTK (SEQ ID NO:132), SAEFNNII (SEQ ID NO:133), FSHR (SEQ ID NO:134), GFSHR (SEQ ID NO:135), EVLGGER (SEQ ID NO:136), GGER (SEQ ID NO:137), FPNYDSR (SEQ ID NO:138), PNYDSR (SEQ ID NO:139), PSAVYR (SEQ ID NO:140), YR, PPR and SGTVDSLDE (SEQ ID NO: 141).

In still other embodiments, Cry1 Ab-specific labeled replacement peptides of the invention comprise amino acid sequence SAEFNNIIPSSQITQIPLTK (SEQ ID NO:21) and produce transition ions consisting of the amino acid sequence PLTK (SEQ ID NO:132) or SAEFNNII (SEQ ID NO: 133).

In some embodiments, the labeled replacement peptides of the invention selectively detect or quantify ecry3.1ab protein and comprise an amino acid sequence selected from the group consisting of: TDVTDYHIDQV (SEQ ID NO:27), AVNELFTSSNQIGLK (SEQ ID NO:28), ITQLPLTK (SEQ ID NO:29), GLDSTTK (SEQ ID NO:30), QCAGIRPYDGR (SEQ ID NO:31) and IEFVPAEVTFEAEYDLER (SEQ ID NO: 32).

In other embodiments, the ecry 3.1ab-specific labeled surrogate peptide produces a transition ion having an amino acid sequence selected from the group consisting of: TDYHIDQV (SEQ ID NO:142), DYYHIDQV (SEQ ID NO:143), TSSNQIGLK (SEQ ID NO:144), SSNQIGLK (SEQ ID NO:145), QLPLTK (SEQ ID NO:146), TQLPLTK (SEQ ID NO:147), DSTTK (SEQ ID NO:148), SSTTK (SEQ ID NO:149), PYDGR (SEQ ID NO:150), DGR, IEF and LER.

In still other embodiments, the eCry 3.1Ab-specific tagged replacement peptide of the invention comprises amino acid sequence TDVTDYHIDQV (SEQ ID NO:27) and generates a transition ion consisting of amino acid sequence TDYHIDQV (SEQ ID NO:142) or DYYHIDQV (SEQ ID NO: 143).

In some embodiments, the labeled replacement peptide selectively detects or quantifies mCry3A protein and comprises an amino acid sequence selected from the group consisting of: ITQLPLVK (SEQ ID NO:33), MTADNNTEALDSSTTK (SEQ ID NO:34), VYIDK (SEQ ID NO:35), and LQSGASVVAGPR (SEQ ID NO: 252).

In other embodiments, the mCry 3A-specific replacement peptide generates a transition ion having an amino acid sequence selected from the group consisting of: QLPLVK (SEQ ID NO:151), TQLPLVK (SEQ ID NO:152), ALDSTTK (SEQ ID NO:153), EALDSSTTK (SEQ ID NO:154), YIDK (SEQ ID NO:155), and IDK.

In still other embodiments, the mCry 3A-specific labeled replacement peptide of the present invention comprises amino acid sequence LQSGASVVAGPR (SEQ ID NO:252) and generates a transition ion consisting of amino acid sequence SGASVVAGPR (SEQ ID NO:253) or SVVAGPR (SEQ ID NO: 254).

In some embodiments, the labeled replacement peptide of the invention selectively detects or quantifies Vip3A protein and comprises an amino acid sequence selected from the group consisting of: DGGISQFIGDK (SEQ ID NO:36), LITTLTCK (SEQ ID NO:37), ELLLATDLSNK (SEQ ID NO:38), FEELTFATETSSK (SEQ ID NO:39), EVLFEK (SEQ ID NO:40), TASELITK (SEQ ID NO:41), DVSEMFTTK (SEQ ID NO:42), LLGLADIDYTSIMNEHLNK (SEQ ID NO:43), IDFTK (SEQ ID NO:44), TDTGGDLTLDEILK (SEQ ID NO:45), DIMNMIFK (SEQ ID NO:46), ALYVHK (SEQ ID NO:47), VNILPTLSNTFSNPNYAK (SEQ ID NO:48), ITSMLSDVIK (SEQ ID NO:49), QNLQLDSFSTYR (SEQ ID NO:50), DSLSEVIYGDMDK (SEQ ID NO:51), MIVEAKPGHALIGFEISNDSITVLK (SEQ ID NO:52), VYFSVSGDANVR (SEQ ID NO:53), NQQLLNDISGK (SEQ ID NO:54), VESSEAEYR (SEQ ID NO:55), YMAK (SEQ ID NO:56), DGSPADILDELTELTELAK (SEQ ID NO:57), YE NO:36), and 8959 (SEQ ID NO: 8958), GKPSIHLK (SEQ ID NO:60), DENTGYIHYEDTNNNLEDYQTINK (SEQ ID NO:61), DNFYIELSQGNNLYGGPIVHFYDVSIK (SEQ ID NO:62), LLCPDQSEQIYYTNNIVFPNEYVITK (SEQ ID NO:63), SQNGDEAWGDNFIILEISPSEK (SEQ ID NO:64), NAYVDHTGGVNGTK (SEQ ID NO:65), LDGVNGSLNDLIAQGNLNTELSK (SEQ ID NO:66), IANEQNQVLNDVNNK (SEQ ID NO:67), YEVTANFYDSSTGEIDLNK (SEQ ID NO:68), QNYALSLQIEYLSK (SEQ ID NO:69), QLQEISIDDK (SEQ ID NO:70), LLSPELINTNNWTSTGSTNISGNTLTLYQGGR (SEQ ID NO:71), YVNEK (SEQ ID NO:72), NYQVDQK (SEQ ID NO:73), and FTTGTDLK (SEQ ID NO: 255).

In other embodiments, the Vip 3A-specifically labeled surrogate peptide produces a transition ion having an amino acid sequence selected from the group consisting of: SQFIGDK (SEQ ID NO:156), GDK, TLTCK (SEQ ID NO:157), TCK, ATDLSNK (SEQ ID NO:158), LATDLSNK (SEQ ID NO:159), TFATETSSK (SEQ ID NO:160), FATETSSK (SEQ ID NO:161), FEK, LFEK (SEQ ID NO:162), SELITK (SEQ ID NO:163), ASELITK (SEQ ID NO:164), SEMFTTK (SEQ ID NO:165), DVS, IMNEHLNK (SEQ ID NO:166), MNLNK (SEQ ID NO:167), DFTK (SEQ ID NO:168), FTK, TLDEILK (SEQ ID NO:169), LTEILK (SEQ ID NO:170), MNMIFK (SEQ ID NO:171), NMIFK (SEQ ID NO:172), YVHK (SEQ ID NO:173), VNHK, SEQ ID NO:174), SMLSR (SEQ ID NO:176), SMLSR (SEQ ID NO:177), VNSTISK (SEQ ID NO:177), NMIFR (SEQ ID NO:178), NMIFNYS NO:177), and SEQ ID NO:177, IYGDMDK (SEQ ID NO:179), VIYGDMDK (SEQ ID NO:180), SNDSITVLK (SEQ ID NO:181), MIV, SGDANVR (SEQ ID NO:182), SVSGDANVR (SEQ ID NO:183), LLNDISGK (SEQ ID NO:184), LNDISGK (SEQ ID NO:185), SSEAEYR (SEQ ID NO:186), ESSEAEYR (SEQ ID NO:187), SGAK (SEQ ID NO:188), MSGAK (SEQ ID NO:189), TELTELAK (SEQ ID NO:190), DGSPADI (SEQ ID NO:191), YEAK (SEQ ID NO:192), EAK, NTMLR (SEQ ID NO:193), AINTR (SEQ ID NO:194), PSDNLK (SEQ ID NO:195), HLK, DYQTINK (SEQ ID NO:196), NK, SEF, FYFY (SEQ ID NO:197), PNEYMLTK (SEQ ID NO:198), PNE TK (SEQ ID NO:199), DHESSEYNKK, SEQ ID NO:200, DHESSENGITK (SEQ ID NO:199), DHISTK: 200, DHESSETK, DHESSETKI (SEQ ID NO:199), and DHISTK), GNLNTELSK (SEQ ID NO:202), NTELSK (SEQ ID NO:203), LNDVNNK (SEQ ID NO:204), NDVNNK (SEQ ID NO:205), YE, DLNK (SEQ ID NO:206), QIEYLSK (SEQ ID NO:207), LQIEYLSK (SEQ ID NO:208), SDK, QEISDK (SEQ ID NO:209), YQGGR (SEQ ID NO:210), TLYQGGR (SEQ ID NO:211), NEK, VNEK (SEQ ID NO:212), DK, and VDK.

In still other embodiments, the Vip 3A-specifically labeled replacement peptide of the invention comprises the amino acid sequence FTTGTDLK (SEQ ID NO:255) and generates a transition ion consisting of the amino acid sequences TGTDLK (SEQ ID NO:256) and LK.

In some embodiments, the labeled replacement peptides of the invention selectively detect or quantify dmesps protein and comprise an amino acid sequence selected from the group consisting of: MAGAEEIVLQPIK (SEQ ID NO:74), FPVEDAK (SEQ ID NO:75), EISGTVK (SEQ ID NO:76), ILLLAALSEGTTVVDNLLNSEDVHYMLGALR (SEQ ID NO:77) and SLTAAVTAAGGNATYVLDGVPR (SEQ ID NO: 257).

In other embodiments, the EPSPS-specific labeled surrogate peptide produces a transition ion having an amino acid sequence selected from the group consisting of: PIK, EIVLQPIK (SEQ ID NO:213), PVEDAK (SEQ ID NO:214), VEDAK (SEQ ID NO:215), SGTVK (SEQ ID NO:216), GTVK (SEQ ID NO:217), ILLLAA (SEQ ID NO:218), and HYMLGALR (SEQ ID NO: 219).

In still other embodiments, the dmesps-specific tagged replacement peptide of the present invention comprises amino acid sequence SLTAAVTAAGGNATYVLDGVPR (SEQ ID NO:257) and produces a transition ion consisting of the amino acid sequences PR and GVPR (SEQ ID NO: 258).

In some embodiments, the labeled replacement peptide of the invention selectively detects or quantifies PAT protein and comprises an amino acid sequence selected from the group consisting of: DFELPAPPRPVRPVTQI (SEQ ID NO:78), LGLGSTLYTHLLK (SEQ ID NO:79), MSPER (SEQ ID NO:80), HGGWHDVGFWQR (SEQ ID NO:81), NAYDWTVESTVYVSHR (SEQ ID NO:82), TEPQTPQEWIDDLER (SEQ ID NO:83), AAGYK (SEQ ID NO:84), YPWLVAEVEGVVAGIAYAGPWK (SEQ ID NO:85) and RPVEIRPATAADMAAVCDIVNHYIETSTVNFR (SEQ ID NO: 86).

In other embodiments, the PAT-specific labeled surrogate peptide produces a transition ion having an amino acid sequence selected from the group consisting of: DFE, DF, YTHLLK (SEQ ID NO:220), THLLK (SEQ ID NO:221), PER, SPER (SEQ ID NO:222), GFWQR (SEQ ID NO:223), VGFWQR (SEQ ID NO:224), STVYVSHR (SEQ ID NO:225), SHR, TEPQT (SEQ ID NO:226), DLER (SEQ ID NO:227), GYK, AGYK (SEQ ID NO:228), GPWK (SEQ ID NO:229), GIAYAGPWK (SEQ ID NO:230), TSTVNFR (SEQ ID NO:231), and NFR.

In still other embodiments, the PAT-specific tagged replacement peptide comprises amino acid sequence LGLGSTLYTHLLK (SEQ ID NO:79) and generates a transition ion consisting of the amino acid sequence YTHLLK (SEQ ID NO:220) or THLLK (SEQ ID NO: 221).

In some embodiments, the labeled replacement peptide of the invention selectively detects or quantifies PMI protein and comprises an amino acid sequence selected from the group consisting of: ENAAGIPMDAAER (SEQ ID NO:87), ALAILK (SEQ ID NO:88), SALDSQQGEPWQTIR (SEQ ID NO:89), GSQQLQLKPGESAFIAANESPVTVK (SEQ ID NO:90), FEAKPANQLLTQPVK (SEQ ID NO:91), STLLGEAVAK (SEQ ID NO:92), LINSVQNYAWGSK (SEQ ID NO:93), HNSEIGFAK (SEQ ID NO:94), VLCAAQPLSIQVHPNK (SEQ ID NO:95), TALTELYGMENPSSQPMAELWMGAHPK (SEQ ID NO:96), LSELFASLLNMQGEEK (SEQ ID NO:97) and QGAELDFPIPVDDFAFSLHDLSDK (SEQ ID NO: 98).

In other embodiments, the PMI-specifically labeled surrogate peptide generates a transition ion having an amino acid sequence selected from the group consisting of: PMDAAER (SEQ ID NO:232), GIPMDAAER (SEQ ID NO:233), AILK (SEQ ID NO:234), LK, PWQTIR (SEQ ID NO:235), GEPWQTIR (SEQ ID NO:236), ANESPVTVK (SEQ ID NO:237), PVTVK (SEQ ID NO:238), LTQPVK (SEQ ID NO:239), PVK, GEAVAK (SEQ ID NO:240), LGEAVAK (SEQ ID NO:241), QNYAWGSK (SEQ ID NO:242), NYAWGSK (SEQ ID NO:243), NSEIGFAK (SEQ ID NO:244), HN, VLCAAQ (SEQ ID NO:245), PNK, WMGAHPK (SEQ ID NO:246), TALTE (SEQ ID NO:247), NMQGEEK (SEQ ID NO:248), LNMQGEEK (SEQ ID NO:249), SLHDLSDK (SEQ ID NO:250), and SEQ ID NO: 251).

In still other embodiments, the PMI-specific replacement peptide comprises the amino acid sequence SALDSQQGEPWQTIR (SEQ ID NO:89) and generates a transition ion consisting of the amino acid sequence PWQTIR (SEQ ID NO:235) or GEPWQTIR (SEQ ID NO: 236).

According to some embodiments, Cry1 Ab-specific labeled alternative peptides of the invention detect and/or quantify a Cry1Ab protein comprising the amino acid sequence of SEQ ID NO: 259. In other embodiments, the Cry1Ab protein is from the transgenic corn event Bt 11.

In some embodiments, the ecry3.1ab specifically labeled replacement peptides of the invention detect and/or quantify the ecry3.1ab protein comprising the amino acid sequence of SEQ ID No. 260. In other embodiments, the ecry3.1ab protein is from transgenic corn event 5307.

According to some embodiments, the mCry 3A-specific labeled replacement peptide of the present invention detects and/or quantifies the mCry3A protein comprising the amino acid sequence of SEQ ID No. 261. In other embodiments, the mCry3A protein is from the transgenic corn event MIR 604.

According to some embodiments, the Vip 3-specific labeled surrogate peptide of the present invention detects and/or quantifies a Vip3Aa protein comprising the amino acid sequence of SEQ ID NO: 262. In other embodiments, the Vip3Aa protein is from the transgenic corn event MIR 162.

According to some embodiments, the dmesps-specific tagged replacement peptides of the present invention detect and/or quantify dmesps protein comprising the amino acid sequence of SEQ ID NO: 263. In other embodiments, the dmesps protein is from the transgenic maize event GA 21.

According to some embodiments, the PAT-specific tagged replacement peptides of the present invention detect and/or quantify the PAT protein comprising the amino acid sequence of SEQ ID NO 264. In other embodiments, the PAT protein is from the transgenic maize event Bt11, 59122, TC1507, DP4114, or T25.

According to some embodiments, the PMI-specifically labeled replacement peptides of the present invention detect and/or quantify PMI proteins comprising the amino acid sequence of SEQ ID NO:265 or SEQ ID NO: 266. In other embodiments, the PMI protein is from the transgenic corn event MIR162, MIR604, 5307, or 3272.

In some embodiments, the labeled replacement peptides of the invention specifically detect or quantify a Cry1Ab protein, ecry3.1ab protein, mCry3A protein, Vip3 protein, dmesps protein, PAT protein, or PMI protein in a mixture of transgenic proteins comprising at least two transgenic proteins selected from the group consisting of: cry1Ab protein, ecry3.1ab protein, mCry3A protein, Vip3A protein, dmesps protein, PAT protein, and PMI protein. In other embodiments, the mixture of transgenic proteins comprises a Cry1Ab protein, an ecry3.1ab protein, an mCry3A protein, a Vip3A protein, a dmesps protein, a PAT protein, and a PMI protein. In still other embodiments, the mixture of transgenic proteins further comprises at least one transgenic protein selected from the group consisting of: Cry1A.105 protein (SEQ ID NO:267), Cry1F protein (SEQ ID NO:268), Cry34 protein (SEQ ID NO:269) and Cry35 protein (SEQ ID NO: 270).

In some embodiments, the labeled replacement peptides of the invention specifically detect or quantify a Cry1Ab protein, ecry3.1ab protein, mCry3A protein, Vip3 protein, dmesps protein, PAT protein, or PMI protein in a protein transgene mixture in a biological sample from a transgenic plant, wherein the transgenic plant is a corn plant, a soybean plant, a cotton plant, a rice plant, a wheat plant, or a canola plant. In other embodiments, the transgenic plant is a maize plant comprising a transgenic event selected from the group consisting of: event Bt11, event 5307, event MIR604, event MIR162, event 3272, and event GA 21. In still other embodiments, the transgenic corn plant further comprises event MON89034, event DP4114, event TC1507, event 59122, or event T25.

In some embodiments, the labeled replacement peptides of the invention specifically detect or quantify Cry1Ab protein, ecry3.1ab protein, mCry3A protein, Vip3 protein, dmesps protein, PAT protein, or PMI protein in a biological sample from leaf tissue, seed, grain, pollen, or root tissue of a transgenic plant. In other embodiments, the leaf tissue, seed, grain, pollen, or root tissue is from a transgenic corn plant comprising one or more of the transgenic corn events Bt11, 5307, MIR604, MIR162, GA21, 3272, 59122, DP4114, TC1507, and T25.

There are many references in the art that suggest many different methods to predict which alternative peptides are optimal for any given target protein, and many have suggested shortcuts to quantify target proteins using mass spectrometry, such as Mead et al 2009 mol cell proteomics 8:696-705 and U.S. patent No. 8,227,252. However, reliance on such prediction methods and shortcuts can lead to confounding results, as unpredictable factors can interfere with mass spectrometry-based assays, resulting in loss of sensitivity and inaccurate quantitative results. At least one major factor is the biological matrix itself. For example, identifying a single transition ion from a surrogate peptide that is as effective as a biological sample from leaves, roots, pollen and seeds of a transgenic plant is highly unpredictable and difficult. Differences in the chemical composition, pH or ionic strength of the matrix may affect proteolysis, peptide stability, aggregation or ionization in MS instruments. Therefore, replacement peptides and specific replacement peptide/transition ion combinations for all relevant substrates (particularly substrates for transgenic plants) must be identified and empirically tested to overcome the unpredictable nature of such assays. The present invention employs a two-step approach in developing mass spectrometry to specifically detect and/or quantify a target transgenic protein, including 1) testing and selecting surrogate peptides from a peptide library derived from a proteolytically cleaved target protein, as well as testing a combination of SIL surrogate peptide and transition ion peptide, and selecting a combination that is capable of specifically detecting and quantifying the target protein in all biological substrates (e.g., biological samples from leaves, roots, pollen or seeds of transgenic plants); and 2) empirically determining sample preparation methods and mass spectrometry conditions suitable for all surrogate peptides and surrogate peptide/transition ion combinations in all biological substrates, including leaves, roots, pollen and seeds of transgenic plants, particularly transgenic corn plants.

Thus, in some embodiments, the invention includes a method of simultaneously detecting and/or quantifying one or more target transgenic proteins in a complex biological sample comprising a mixture of the target transgenic protein and a non-transgenic protein from a transgenic plant, wherein the method comprises the steps of: a) obtaining a biological sample from the transgenic plant; b) extracting proteins from said biological sample to obtain an extract comprising a mixture of proteins; c) reducing the amount of non-transgenic insoluble protein in the extract of step b to obtain a concentrated extract of soluble protein; d) digesting the soluble proteins in the extract of step c to obtain an extract comprising peptide fragments, wherein the peptide fragments comprise at least one unlabeled surrogate peptide specific for each target transgenic protein; e) concentrating the peptide fragments in the extract of step d; f) adding one or more labeled replacement peptides of the invention, wherein each labeled replacement peptide has the same amino acid sequence as each unlabeled replacement peptide derived from the target transgenic protein, and wherein the number of labeled replacement peptides added is equal to the number of target transgenic proteins in the mixture; g) concentrating the unlabeled surrogate peptide and the labeled surrogate peptide by reducing the amount of non-surrogate peptide in the mixture; h) decomposing the peptide fragment mixture of step g by liquid chromatography; i) analyzing the peptide fragment mixture resulting from step h by mass spectrometry, wherein detection of a transition ion fragment of an unlabeled surrogate peptide indicates the presence of the target transgenic protein from which the surrogate peptide was derived; and, optionally, j) calculating the amount of the target transgenic protein in the biological sample by comparing the mass spectral signal generated by the transition ion fragment of step i with the mass spectral signal generated by the transition ion of the labeled surrogate peptide.

In some embodiments, the target transgenic protein detected and/or quantified by the above-described methods is a Cry1Ab protein, an ecry3.1ab protein, a mCry3A protein, a Vip3 protein, a double mutant 5-enolpyruvylshikimate-3-phosphate synthase (dmesps) protein, a glufosinate acetyltransferase (PAT) protein, or a phosphomannose isomerase (PMI) protein.

In other embodiments encompassed by the methods of the invention, the target transgenic protein is Cry1Ab and the labeled replacement peptide comprises amino acid sequence SAEFNNIIPSSQITQIPLTK (SEQ ID NO:21) and produces a transition ion consisting of the amino acid sequence PLTK (SEQ ID NO:132) or SAEFNNII (SEQ ID NO: 133). In still other embodiments, the Cry1Ab target protein is quantified by comparing the mass spectral signals generated by unlabeled and labeled transition ions consisting of the amino acid sequence PLTK (SEQ ID NO: 132).

In other embodiments encompassed by the methods of the invention, the target transgenic protein is eCry3.1Ab and the tagged replacement peptide comprises amino acid sequence TDVTDYHIDQV (SEQ ID NO:27) and generates a transition ion consisting of the amino acid sequence TDYHIDQV (SEQ ID NO:142) or DYYHIDQV (SEQ ID NO: 143). In still other embodiments, the eCry3.1Ab target protein is quantified by comparing the mass spectral signals generated by unlabeled and labeled transition ions consisting of the amino acid sequence TDYHIDQV (SEQ ID NO: 142).

In other embodiments encompassed by the methods of the present invention, the target transgenic protein is mCry3A and the tagged replacement peptide comprises amino acid sequence LQSGASVVAGPR (SEQ ID NO:252) and produces a transition ion consisting of amino acid sequence SGASVVAGPR (SEQ ID NO:253) or SVVAGPR (SEQ ID NO: 254). In still other embodiments, the mCry3A target protein is quantified by comparing the mass spectral signals generated by unlabeled and labeled transition ions consisting of amino acid sequence SGASVVAGPR (SEQ ID NO: 253).

In other embodiments encompassed by the methods of the invention, the target transgenic protein is Vip3A and the tagged replacement peptide comprises the amino acid sequence FTTGTDLK (SEQ ID NO:255) and generates a transition ion consisting of the amino acid sequence TGTDLK (SEQ ID NO:256) or LK. In still other embodiments, the Vip3A target protein is quantified by comparing the mass spectral signals generated by unlabeled and labeled transition ions consisting of the amino acid sequence TGTDLK (SEQ ID NO: 256).

In other embodiments encompassed by the methods of the invention, the target transgene protein is dmesps, and the tagged replacement peptide comprises amino acid sequence SLTAAVTAAGGNATYVLDGVPR (SEQ ID NO:257) and produces a transition ion consisting of amino acid sequence PR or GVPR (SEQ ID NO: 258). In still other embodiments, the ecry3.1ab target protein is quantified by comparing the mass spectral signals generated by unlabeled and labeled transition ions consisting of the amino acid sequence PR.

In other embodiments encompassed by the methods of the invention, the target transgenic protein is PAT and the tagged replacement peptide comprises amino acid sequence LGLGSTLYTHLLK (SEQ ID NO:79) and generates a transition ion consisting of the amino acid sequence YTHLLK (SEQ ID NO:220) or THLLK (SEQ ID NO: 221). In still other embodiments, the PAT target protein is quantified by comparing the mass spectral signals generated by unlabeled and labeled transition ions consisting of the amino acid sequence YTHLLK (SEQ ID NO: 220).

In other embodiments encompassed by the methods of the invention, the target transgenic protein is PMI and the replacement peptide for the marker comprises amino acid sequence SALDSQQGEPWQTIR (SEQ ID NO:89) and generates a transition ion consisting of the amino acid sequence PWQTIR (SEQ ID NO:235) or GEPWQTIR (SEQ ID NO: 236). In still other embodiments, the PMI target protein is quantified by comparing the mass spectral signals generated by unlabeled and labeled transition ions consisting of the amino acid sequence PWQTIR (SEQ ID NO: 235).

In other embodiments, the invention includes systems for high throughput detection or quantification of transgenic target proteins. Such systems comprise a cassette of pre-designed labeled surrogate peptides specific for the transgenic target protein; and one or more mass spectrometers. In one aspect of this embodiment, the cassette comprises a labeled surrogate peptide specific for a target protein selected from the group consisting of: cry1Ab, ecry3.1ab, mCry3A, Vip3, dmesps, PAT, and PMI. In other aspects of this embodiment, the labeled replacement peptide comprises any one of SEQ ID NOs 1-98. In other aspects of this embodiment, the labeled surrogate peptide generates one or more transition ions comprising a peptide sequence selected from the group consisting of at least one of SEQ ID NOs 99-251, SEQ ID NOs 254, 255, 256, peptide PIR, TY, VW, HR, ISR, LEG, QFR, YR, PPR, DGR, IEF, LER, IDK, GDK, TCK, FEK, DVS, FTK, HK, VNI, MIV, EAK, HLK, NK, DNF, LLC, NAY, YE, SDK, NEK, DK, VDK, PIK, DFE, DF, PER, SHR, GYK, NFR, LK, PVK, HN, PNK, and PR.

The following specific examples are included to illustrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Examples of the invention

Example 1 alternative peptide selection

MRM-based assays rely on the selection of a predetermined set of peptides and on the specific fragmentation/transition ion of each selected surrogate peptide. Several criteria are required to select the appropriate surrogate or signature peptide. First, the proteins that make up the targeted protein cassette must be selected. Second, for each target protein, those peptides that exhibit good mass spectral response and uniquely identify the target protein or specific modifications (i.e., post-translational modifications) thereof must be identified. Third, for each peptide of the appropriate mass spectrum, those transition ions that provide the best signal intensity and uniquely distinguish the surrogate peptide from the other peptide species present in the sample must be identified. These criteria are crucial for performing MRM-based assays.

Replacement peptides from seven transgenic proteins Cry1Ab, ecry3.1ab, mCry3A, Vip3A, dmesps, PAT, and PMI were identified and selected for MRM-based assays. The MRM assay was developed using microbially produced proteins, which were digested with trypsin. The proteins produced by the microorganisms were reconstituted separately with water. Trifluoroethanol (TFE) was then added to an aliquot of each protein, followed by 100mM ammonium bicarbonate and trypsin (1:10(w: w) enzyme: protein ratio). The sample was digested overnight at about 37 ℃ and then about 0.05M tris (2-carboxyethyl) phosphine (TCEP) was added. Each protein was aliquoted to form a pool with a final concentration of about 200 pmol/. mu.L. The peptide mixture was used to develop the MRM assay on a QTRAP 6500 mass spectrometer (AB Sciex LLC), framingham, massachusetts, usa. MS/MS was monitored using the selected reactions to determine the optimal two transitions (combination of peptide surrogate and fragment ion mass to charge ratio (m/z) monitored by the mass spectrometer) for each peptide. The MS/MS method was developed by calculating the characteristic mass of the doubly and triply charged peptide ions and the first and second y fragment ions (whose m/z is greater than [ m/z (alternative) +20Da ]) for each peptide. If these calculated transitions are observed during the MRM scan, the instrument will automatically switch to MS/MS mode and obtain a complete MS/MS spectrum of the surrogate peptide ion. For each peptide obtained, the two strongest fragment ions (only b or y fragment ions) in the MS/MS spectrum and their elution times were determined. The Collision Energy (CE) is then optimized for each selected transition. The MRM assay developed was used for the analysis of calibration curve samples.

This MRM assay targets 193 protein-type peptides (proteotypic peptides) from 7 transgenic proteins. Of these, 111 peptides are unique to these 7 proteins and do not overlap with known zeins. Table 1 lists the characteristics of the surrogate peptides and transition ions for each target protein, including the amino acid sequence (including sequence listing identifiers for peptides comprising at least four amino acids), monoisotopic mass, characteristic charge state, characteristic m/z, and product transition m/z. Unique replacement peptides for all seven proteins were identified; cry1Ab (26), ecry3.1ab (6), mCry3A (4), Vip3Aa20(39), dmesps (5), PAT (9), and PMI (12). These replacement peptides from Cry1Ab, ecry3.1ab, mCry3A, Vip3A, dmesps, PAT, and PMI were identified for use in determining absolute or relative amounts of Cry1Ab, ecry3.1ab, mCry3A, Vip3A, dmesps, PAT, and PMI transgene proteins. Each of these peptides or combinations of peptides listed in table 1 was detected in lysates by mass spectrometry and is a potential candidate for quantification of Cry1Ab, ecry3.1ab, mCry3A, Vip3A, dmesps, PAT, and PMI in MRM-based assays.

TABLE 1 characteristics of the surrogate peptides and transition ions.

Having identified a number of potential replacement peptides for the target proteins Cry1Ab, ecry3.1ab, mCry3A, Vip3A, dmesps, PAT, and PMI, individual replacement peptides are further selected based on transition ions that provide the best signal strength and have the ability to distinguish the target replacement peptide from other species present in the biological sample matrix (e.g., corn leaf, root, pollen, or grain (seed)). This includes both matrix interference (i.e., matrix interference is one or more specific components in the matrix detected at or near the peptide of interest) and potential carryover (i.e., carryover is a result of previously injected samples eluting in subsequent analyses due to the chemical/physical properties of the sample analysis system, or both). These optimized transitions of the cassette containing the individual surrogate peptides make up the overall MRM assay. In the present disclosure, those replacement peptides from Cry1Ab, ecry3.1ab, mCry3A, Vip3A, dmesps, PAT and PMI that provided the highest sensitivity (the strongest fragment) and had the desired specificity were further selected to constitute a cassette of replacement peptides for quantification of the seven targeted proteins. Table 2 lists preferred replacement peptides for each of the target proteins Cry1Ab, ecry3.1ab, mCry3A, Vip3Aa20 dmesps, PAT and PMI, as well as the corresponding Stable Isotope Labeled (SIL) peptides. The cassette of surrogate peptides comprises one or more of the peptides to be monitored and/or quantified simultaneously. This cassette with replacement peptides for the specific fragmentation/transition ion of each peptide can be used in MRM assays to quantify the corresponding target protein.

TABLE 2 detection of replacement peptides and SIL peptides of the target protein specifically.

Example 2-assay for detection of transgenic proteins in transgenic plant tissues

Development of sensitive methods for direct detection of target proteins is highly desirable for quantitative evaluation in biological substrates such as tissues of transgenic plants, e.g., leaf, grain, root and pollen tissues. Multiple Reaction Monitoring (MRM) mass spectrometry has become a promising platform to quantify multiple proteins in a given sample by Liquid Chromatography (LC) in combination with tandem mass spectrometry (MS /). MRM assays utilize sequence-specific tandem MS fragmentation of proteolytic peptides, providing highly selective and specific measurements for different target proteins. Despite these advances, accurate quantitative measurements of low abundance proteins or proteins with specific physicochemical properties that affect isolation remain challenging.

MRM measurements are typically performed on a triple quadrupole mass spectrometer, although the method may also be applied in ion trap instruments where MS/MS data is acquired on fragment ions within a defined mass range or over the entire mass range after fragmentation of the characteristic ions. A series of transitions (feature/fragment ion m/z pairs) combined with the retention time of the targeting peptide can constitute an MRM assay. To achieve an optimal MRM assay, (1) the target protein/peptide needs to be selected; (2) the surrogate peptide must produce good MS and MS/MS signals; (3) each selected peptide fragment ion must provide the best signal intensity and distinguish the target peptide from other peptide species present in the complex biological sample. Overall, the surrogate peptide and fragment ions provide high specificity for peptide selection, since only the desired transition is recorded and other signals present in the sample are ignored.

A common misconception in the art is that MRM assays can ensure specificity and sensitivity, sample preparation can be simplified or even omitted, and no or very few chromatographic separations are required. However, contrary to this false recognition, MRM assays tend to be greatly affected by the complexity of the sample, thereby reducing the sensitivity of a particular target peptide. This specificity and sensitivity may be affected by matrix effects such as differences between leaves, pollen, roots, stems and lead to ion suppression, which occurs during MS analysis. Generally, most charged or ionizable molecules, such as salts, chaotropes, detergents, polymers, all non-volatile ionic compounds, interfere with ionization of the desired analyte (i.e., peptide/protein), thereby competing and resulting in signal suppression and/or increased background noise. Ion suppression can negatively impact several analytical parameters, such as detection capability, accuracy and precision. Therefore, to overcome all these drawbacks in the art of MRM mass spectrometry, it is desirable to develop a method for efficiently extracting a target protein from a complex biological sample (e.g., a transgenic plant sample) to enrich for the target protein and/or eliminate interference that may reduce the ionic strength of the target protein/peptide and affect the reproducibility and accuracy of the assay.

Overall, improving sample preparation may be the most effective way to reduce matrix effects and avoid ion suppression. The method enables the enrichment of selected target proteins and peptides without the ability to concentrate interference, allowing accurate and precise quantitation at low target protein concentrations.

MRM-based assays using cassettes of replacement peptides (from table 1 or table 2) measure Cry1Ab, ecry3.1ab, mCry3A, Vip3A, dmesps, PAT, and PMI in different transgenic events containing at least one of the seven proteins (table 4). Transgenic events evaluated in this study were as follows: bt11(Cry1Ab and PAT); 5307(ecry3.1ab and PMI); MIR604(mCry3A and PMI); MIR162(Vip3Aa20 and PMI) and GA21 (dmesps).

Tissue extraction-12-15 mg of freeze-dried tissue (leaves, roots, pollen, grain and whole plants) was placed in 2 μ L lysis matrix a FastPrep tube (MP bio-medical company, san ana, ca). Then 1.0-1.5. mu.L (w/v) of PBS containing 0.1% RapidGest was added. Samples were then taken at ambient temperature in a FastPrep-24 tissue homogenizer (MP biomedical corporation, san anna, ca) with lysis matrix a (garnet matrix and 1/4 "ceramic beads) for 1 cycle (40s, speed setting 6). Protein extraction from selected tissues in 50. mu.l extraction buffer (6M Urea, 2M Thiourea, 5mM EDTA, 0.1M HEPES) per mg of lyophilized tissue

After centrifugation-tissue extraction, the sample was centrifuged at 15,000g for about 5min at4 ℃. This step removes insoluble proteins, e.g., histones and actins, thereby reducing the complexity of the extract prior to digestion. Therefore, only soluble proteins can enter the enzymatic digestion step.

Trypsinization-the total protein concentration of the supernatant in this centrifugation step was adjusted to about 0.2 μ g/μ l by dilution in homogenization buffer. Equivalent to 30. mu.g of protein was transferred to the well plate. A volume of trifluoroethanol was added to the sample and incubated at room temperature for about 30min with low speed shaking. Four volumes of 100mM ammonium bicarbonate were added. Then about 12. mu.l trypsin (0.1. mu.g/. mu.l) was added. The samples were incubated overnight at 37 ℃. The sample was then quenched with 20% formic acid (final concentration 1%). Then 20. mu.l of the stable isotope labeled peptide was added.

Centrifugation-the sample from the previous step was centrifuged at 15,000g for about 5min at4 ℃.

After desalting by MCX-centrifugation, the sample was desalted. This step is carried out on an ion exchange column. The desalting step concentrates the peptide of interest by discarding the non-peptide of interest from the wash tank. In addition to removing the non-peptide of interest, this step also removes salts and small molecules that may interfere with ionization and detection of the surrogate peptide of interest. Concentrating the peptide of interest and removing interfering salts and small molecules may improve the sensitivity of the MRM assay of the invention compared to other methods known in the art.

QTRAP-MRM-MRM analysis was performed using a Halo Peptide ES-C18 column using QTRAP 6500 in combination with Nanoacity UPLC. The flow rate was about 18. mu.l/min. Solvent a was about 97/3 water/DMSO + 0.2% Formic Acid (FA), and solvent B was about 97/3 acetonitrile (CAN)/DMSO + 0.2% FA. During the analysis, the temperature of the autosampler was maintained at about 4 ℃. A total of 8. mu.l of sample was injected onto the column, which was kept at ambient temperature.

Data analysis-data collection was performed using Analyst software (eboothi, ontario, canada) and data analysis was performed using Multiquant software (eboothi).

To determine the level of detection (LOD) of the target transgenic protein using the preferred labeled replacement peptides of the invention, all seven target proteins Cry1Ab, ecry3.1ab, mCry3A, Vip3A, dmesps, PAT, and PMI were mixed together and added to leaf, root, grain, and pollen tissues of non-transgenic corn plants. Tables 3-6 show the level of detection (LOD) of the target protein by the MRM and indicate that each labeled replacement peptide and its resulting transition ion can selectively detect and quantify the target protein when the target protein is accompanied by other transgenic and non-transgenic proteins in all plant substrates. Good linearity was obtained for each of the preferred alternative peptides of table 2 (r ═ 0.988-0.998). The LOD of each surrogate peptide was below the range determined by ELISA, indicating that the compositions and methods of the invention are equal to or superior to current standards for quantifying transgenic proteins in plants.

TABLE 3 LOD of target protein in maize leaf matrix. (LOD ═ fmol/. mu.g total protein)

TABLE 4 LOD of target protein in corn grain matrix. (LOD ═ fmol/. mu.g total protein)

TABLE 5 LOD of target protein in corn root matrix. (LOD ═ fmol/. mu.g total protein)

TABLE 6 LOD of target protein in maize pollen matrix. (LOD ═ fmol/. mu.g total protein)

The preferred labeled surrogate peptides (table 2) and their transition ions were then tested to determine their ability to specifically detect a target protein in leaf, grain, root and pollen tissues from a transgenic corn plant comprising a transgenic event selected from the group consisting of: bt11 (containing Cry1Ab and PAT), 5307 (containing ecry3.1ab and PMI), MIR604 (containing mCry3A and PMI), MIR162 (containing Vip3A and PMI), and GA21 (containing dmesps). Each of the seven preferred surrogate peptides was tested for each transgenic event. Table 7 shows the results of quantification of the target protein. This result indicates that the Cry1Ab and PAT replacement peptides and labeled replacement peptides are capable of detecting and/or quantifying Cry1Ab and PAT in leaves, grains, roots and pollen from transgenic corn plants comprising event Bt 11. For the plants tested, the Cry1Ab protein was lower than the LOD in pollen (see Table 5) tissues, while the PAT protein was lower than the LOD in grains and pollen (see tables 4 and 5). The ecry3.1ab and PMI replacement peptides and labeled replacement peptides were able to detect ecry3.1ab and PMI from leaves, grain, roots, and pollen of transgenic corn plants comprising event 5307. For the plants tested, the ecry3.1ab protein was lower than the LOD in pollen (see table 5). The mCry3A and PMI replacement peptide and labeled replacement peptide were able to detect mCry3A and PMI protein from leaves, grains, roots, and pollen of transgenic corn plants comprising event MIR 604. For the plants tested, the mCry3A protein was lower than the LOD in pollen (see table 5). The Vip3Aa20 and PMI replacement peptides and labeled replacement peptides were able to detect Vip3Aa20 and PMI proteins from leaves, grains, roots, and pollen of transgenic corn plants comprising event MIR 162. The dmesps replacement peptide and labeled replacement peptide are capable of detecting dmesps protein in leaves, grains, roots, and pollen from a transgenic corn plant comprising event GA 21.

To further characterize the ability of preferred tagged replacement peptides of the invention, the above assays were performed on a breeding stack expressing all seven proteins (Cry1Ab, ecry3.1ab, mCry3A, Vip3A, dmesps, PAT, and PMI). This result indicates that LC-SRM can simultaneously detect and quantify all seven proteins contained in this breeding stack.

The substitution peptides and labeled substitution peptides listed in table 1 and/or table 2 enable detection and/or quantification of the target proteins of the invention. Each of these peptides or combinations of these peptides are candidates for use in quantitative MRM assays for the target protein.

TABLE 7 detection and quantification of target proteins in transgenic plants.

nd is not measured

While the invention has been described in connection with specific embodiments thereof, it will be understood that the apparatus of the invention is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains: which are within the known or customary practice in the art to which this invention pertains and which may be applied to the key features hereinbefore set forth and to the scope of the appended claims.

All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All such publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Sequence listing

<110> Syngenta Participations AG

Young, Scott

Sessler, Richard

Graser, Gerson

Guilbaud, Rudolf

Schirm, Michael

Isabelle, Maxim

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<223> engineered hybrid toxins

<400> 27

Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val

1 5 10

<210> 28

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> engineered hybrid toxins

<400> 28

Ala Val Asn Glu Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu Lys

1 5 10 15

<210> 29

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> engineered hybrid toxins

<400> 29

Ile Thr Gln Leu Pro Leu Thr Lys

1 5

<210> 30

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> engineered hybrid toxins

<400> 30

Gly Leu Asp Ser Ser Thr Thr Lys

1 5

<210> 31

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> engineered hybrid toxins

<400> 31

Gln Cys Ala Gly Ile Arg Pro Tyr Asp Gly Arg

1 5 10

<210> 32

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> engineered hybrid toxins

<400> 32

Ile Glu Phe Val Pro Ala Glu Val Thr Phe Glu Ala Glu Tyr Asp Leu

1 5 10 15

Glu Arg

<210> 33

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> mutant Cry3A

<400> 33

Ile Thr Gln Leu Pro Leu Val Lys

1 5

<210> 34

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> mutant Cry3A

<400> 34

Met Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys

1 5 10 15

<210> 35

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> mutant Cry3A

<400> 35

Val Tyr Ile Asp Lys

1 5

<210> 36

<211> 11

<212> PRT

<213> Bacillus thuringiensis

<400> 36

Asp Gly Gly Ile Ser Gln Phe Ile Gly Asp Lys

1 5 10

<210> 37

<211> 7

<212> PRT

<213> Bacillus thuringiensis

<400> 37

Leu Ile Thr Leu Thr Cys Lys

1 5

<210> 38

<211> 11

<212> PRT

<213> Bacillus thuringiensis

<400> 38

Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys

1 5 10

<210> 39

<211> 13

<212> PRT

<213> Bacillus thuringiensis

<400> 39

Phe Glu Glu Leu Thr Phe Ala Thr Glu Thr Ser Ser Lys

1 5 10

<210> 40

<211> 6

<212> PRT

<213> Bacillus thuringiensis

<400> 40

Glu Val Leu Phe Glu Lys

1 5

<210> 41

<211> 8

<212> PRT

<213> Bacillus thuringiensis

<400> 41

Thr Ala Ser Glu Leu Ile Thr Lys

1 5

<210> 42

<211> 9

<212> PRT

<213> Bacillus thuringiensis

<400> 42

Asp Val Ser Glu Met Phe Thr Thr Lys

1 5

<210> 43

<211> 19

<212> PRT

<213> Bacillus thuringiensis

<400> 43

Leu Leu Gly Leu Ala Asp Ile Asp Tyr Thr Ser Ile Met Asn Glu His

1 5 10 15

Leu Asn Lys

<210> 44

<211> 5

<212> PRT

<213> Bacillus thuringiensis

<400> 44

Ile Asp Phe Thr Lys

1 5

<210> 45

<211> 14

<212> PRT

<213> Bacillus thuringiensis

<400> 45

Thr Asp Thr Gly Gly Asp Leu Thr Leu Asp Glu Ile Leu Lys

1 5 10

<210> 46

<211> 8

<212> PRT

<213> Bacillus thuringiensis

<400> 46

Asp Ile Met Asn Met Ile Phe Lys

1 5

<210> 47

<211> 6

<212> PRT

<213> Bacillus thuringiensis

<400> 47

Ala Leu Tyr Val His Lys

1 5

<210> 48

<211> 18

<212> PRT

<213> Bacillus thuringiensis

<400> 48

Val Asn Ile Leu Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro Asn Tyr

1 5 10 15

Ala Lys

<210> 49

<211> 10

<212> PRT

<213> Bacillus thuringiensis

<400> 49

Ile Thr Ser Met Leu Ser Asp Val Ile Lys

1 5 10

<210> 50

<211> 12

<212> PRT

<213> Bacillus thuringiensis

<400> 50

Gln Asn Leu Gln Leu Asp Ser Phe Ser Thr Tyr Arg

1 5 10

<210> 51

<211> 13

<212> PRT

<213> Bacillus thuringiensis

<400> 51

Asp Ser Leu Ser Glu Val Ile Tyr Gly Asp Met Asp Lys

1 5 10

<210> 52

<211> 25

<212> PRT

<213> Bacillus thuringiensis

<400> 52

Met Ile Val Glu Ala Lys Pro Gly His Ala Leu Ile Gly Phe Glu Ile

1 5 10 15

Ser Asn Asp Ser Ile Thr Val Leu Lys

20 25

<210> 53

<211> 12

<212> PRT

<213> Bacillus thuringiensis

<400> 53

Val Tyr Phe Ser Val Ser Gly Asp Ala Asn Val Arg

1 5 10

<210> 54

<211> 11

<212> PRT

<213> Bacillus thuringiensis

<400> 54

Asn Gln Gln Leu Leu Asn Asp Ile Ser Gly Lys

1 5 10

<210> 55

<211> 9

<212> PRT

<213> Bacillus thuringiensis

<400> 55

Val Glu Ser Ser Glu Ala Glu Tyr Arg

1 5

<210> 56

<211> 6

<212> PRT

<213> Bacillus thuringiensis

<400> 56

Tyr Met Ser Gly Ala Lys

1 5

<210> 57

<211> 19

<212> PRT

<213> Bacillus thuringiensis

<400> 57

Asp Gly Ser Pro Ala Asp Ile Leu Asp Glu Leu Thr Glu Leu Thr Glu

1 5 10 15

Leu Ala Lys

<210> 58

<211> 5

<212> PRT

<213> Bacillus thuringiensis

<400> 58

Val Tyr Glu Ala Lys

1 5

<210> 59

<211> 9

<212> PRT

<213> Bacillus thuringiensis

<400> 59

Leu Asp Ala Ile Asn Thr Met Leu Arg

1 5

<210> 60

<211> 8

<212> PRT

<213> Bacillus thuringiensis

<400> 60

Gly Lys Pro Ser Ile His Leu Lys

1 5

<210> 61

<211> 24

<212> PRT

<213> Bacillus thuringiensis

<400> 61

Asp Glu Asn Thr Gly Tyr Ile His Tyr Glu Asp Thr Asn Asn Asn Leu

1 5 10 15

Glu Asp Tyr Gln Thr Ile Asn Lys

20

<210> 62

<211> 27

<212> PRT

<213> Bacillus thuringiensis

<400> 62

Asp Asn Phe Tyr Ile Glu Leu Ser Gln Gly Asn Asn Leu Tyr Gly Gly

1 5 10 15

Pro Ile Val His Phe Tyr Asp Val Ser Ile Lys

20 25

<210> 63

<211> 26

<212> PRT

<213> Bacillus thuringiensis

<400> 63

Leu Leu Cys Pro Asp Gln Ser Glu Gln Ile Tyr Tyr Thr Asn Asn Ile

1 5 10 15

Val Phe Pro Asn Glu Tyr Val Ile Thr Lys

20 25

<210> 64

<211> 22

<212> PRT

<213> Bacillus thuringiensis

<400> 64

Ser Gln Asn Gly Asp Glu Ala Trp Gly Asp Asn Phe Ile Ile Leu Glu

1 5 10 15

Ile Ser Pro Ser Glu Lys

20

<210> 65

<211> 14

<212> PRT

<213> Bacillus thuringiensis

<400> 65

Asn Ala Tyr Val Asp His Thr Gly Gly Val Asn Gly Thr Lys

1 5 10

<210> 66

<211> 23

<212> PRT

<213> Bacillus thuringiensis

<400> 66

Leu Asp Gly Val Asn Gly Ser Leu Asn Asp Leu Ile Ala Gln Gly Asn

1 5 10 15

Leu Asn Thr Glu Leu Ser Lys

20

<210> 67

<211> 15

<212> PRT

<213> Bacillus thuringiensis

<400> 67

Ile Ala Asn Glu Gln Asn Gln Val Leu Asn Asp Val Asn Asn Lys

1 5 10 15

<210> 68

<211> 19

<212> PRT

<213> Bacillus thuringiensis

<400> 68

Tyr Glu Val Thr Ala Asn Phe Tyr Asp Ser Ser Thr Gly Glu Ile Asp

1 5 10 15

Leu Asn Lys

<210> 69

<211> 14

<212> PRT

<213> Bacillus thuringiensis

<400> 69

Gln Asn Tyr Ala Leu Ser Leu Gln Ile Glu Tyr Leu Ser Lys

1 5 10

<210> 70

<211> 8

<212> PRT

<213> Bacillus thuringiensis

<400> 70

Gln Leu Gln Glu Ile Ser Asp Lys

1 5

<210> 71

<211> 32

<212> PRT

<213> Bacillus thuringiensis

<400> 71

Leu Leu Ser Pro Glu Leu Ile Asn Thr Asn Asn Trp Thr Ser Thr Gly

1 5 10 15

Ser Thr Asn Ile Ser Gly Asn Thr Leu Thr Leu Tyr Gln Gly Gly Arg

20 25 30

<210> 72

<211> 5

<212> PRT

<213> Bacillus thuringiensis

<400> 72

Tyr Val Asn Glu Lys

1 5

<210> 73

<211> 7

<212> PRT

<213> Bacillus thuringiensis

<400> 73

Gln Asn Tyr Gln Val Asp Lys

1 5

<210> 74

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> mutant EPSPS

<400> 74

Met Ala Gly Ala Glu Glu Ile Val Leu Gln Pro Ile Lys

1 5 10

<210> 75

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> mutant EPSPS

<400> 75

Phe Pro Val Glu Asp Ala Lys

1 5

<210> 76

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> mutant EPSPS

<400> 76

Glu Ile Ser Gly Thr Val Lys

1 5

<210> 77

<211> 31

<212> PRT

<213> Artificial sequence

<220>

<223> mutant EPSPS

<400> 77

Ile Leu Leu Leu Ala Ala Leu Ser Glu Gly Thr Thr Val Val Asp Asn

1 5 10 15

Leu Leu Asn Ser Glu Asp Val His Tyr Met Leu Gly Ala Leu Arg

20 25 30

<210> 78

<211> 17

<212> PRT

<213> Streptomyces viridochromogenes

<400> 78

Asp Phe Glu Leu Pro Ala Pro Pro Arg Pro Val Arg Pro Val Thr Gln

1 5 10 15

Ile

<210> 79

<211> 13

<212> PRT

<213> Streptomyces viridochromogenes

<400> 79

Leu Gly Leu Gly Ser Thr Leu Tyr Thr His Leu Leu Lys

1 5 10

<210> 80

<211> 5

<212> PRT

<213> Streptomyces viridochromogenes

<400> 80

Met Ser Pro Glu Arg

1 5

<210> 81

<211> 12

<212> PRT

<213> Streptomyces viridochromogenes

<400> 81

His Gly Gly Trp His Asp Val Gly Phe Trp Gln Arg

1 5 10

<210> 82

<211> 16

<212> PRT

<213> Streptomyces viridochromogenes

<400> 82

Asn Ala Tyr Asp Trp Thr Val Glu Ser Thr Val Tyr Val Ser His Arg

1 5 10 15

<210> 83

<211> 15

<212> PRT

<213> Streptomyces viridochromogenes

<400> 83

Thr Glu Pro Gln Thr Pro Gln Glu Trp Ile Asp Asp Leu Glu Arg

1 5 10 15

<210> 84

<211> 5

<212> PRT

<213> Streptomyces viridochromogenes

<400> 84

Ala Ala Gly Tyr Lys

1 5

<210> 85

<211> 22

<212> PRT

<213> Streptomyces viridochromogenes

<400> 85

Tyr Pro Trp Leu Val Ala Glu Val Glu Gly Val Val Ala Gly Ile Ala

1 5 10 15

Tyr Ala Gly Pro Trp Lys

20

<210> 86

<211> 32

<212> PRT

<213> Streptomyces viridochromogenes

<400> 86

Arg Pro Val Glu Ile Arg Pro Ala Thr Ala Ala Asp Met Ala Ala Val

1 5 10 15

Cys Asp Ile Val Asn His Tyr Ile Glu Thr Ser Thr Val Asn Phe Arg

20 25 30

<210> 87

<211> 13

<212> PRT

<213> Escherichia coli

<400> 87

Glu Asn Ala Ala Gly Ile Pro Met Asp Ala Ala Glu Arg

1 5 10

<210> 88

<211> 6

<212> PRT

<213> Escherichia coli

<400> 88

Ala Leu Ala Ile Leu Lys

1 5

<210> 89

<211> 15

<212> PRT

<213> Escherichia coli

<400> 89

Ser Ala Leu Asp Ser Gln Gln Gly Glu Pro Trp Gln Thr Ile Arg

1 5 10 15

<210> 90

<211> 25

<212> PRT

<213> Escherichia coli

<400> 90

Gly Ser Gln Gln Leu Gln Leu Lys Pro Gly Glu Ser Ala Phe Ile Ala

1 5 10 15

Ala Asn Glu Ser Pro Val Thr Val Lys

20 25

<210> 91

<211> 15

<212> PRT

<213> Escherichia coli

<400> 91

Phe Glu Ala Lys Pro Ala Asn Gln Leu Leu Thr Gln Pro Val Lys

1 5 10 15

<210> 92

<211> 10

<212> PRT

<213> Escherichia coli

<400> 92

Ser Thr Leu Leu Gly Glu Ala Val Ala Lys

1 5 10

<210> 93

<211> 13

<212> PRT

<213> Escherichia coli

<400> 93

Leu Ile Asn Ser Val Gln Asn Tyr Ala Trp Gly Ser Lys

1 5 10

<210> 94

<211> 9

<212> PRT

<213> Escherichia coli

<400> 94

His Asn Ser Glu Ile Gly Phe Ala Lys

1 5

<210> 95

<211> 16

<212> PRT

<213> Escherichia coli

<400> 95

Val Leu Cys Ala Ala Gln Pro Leu Ser Ile Gln Val His Pro Asn Lys

1 5 10 15

<210> 96

<211> 27

<212> PRT

<213> Escherichia coli

<400> 96

Thr Ala Leu Thr Glu Leu Tyr Gly Met Glu Asn Pro Ser Ser Gln Pro

1 5 10 15

Met Ala Glu Leu Trp Met Gly Ala His Pro Lys

20 25

<210> 97

<211> 16

<212> PRT

<213> Escherichia coli

<400> 97

Leu Ser Glu Leu Phe Ala Ser Leu Leu Asn Met Gln Gly Glu Glu Lys

1 5 10 15

<210> 98

<211> 24

<212> PRT

<213> Escherichia coli

<400> 98

Gln Gly Ala Glu Leu Asp Phe Pro Ile Pro Val Asp Asp Phe Ala Phe

1 5 10 15

Ser Leu His Asp Leu Ser Asp Lys

20

<210> 99

<211> 7

<212> PRT

<213> Bacillus thuringiensis

<400> 99

Gly Ile Glu Gly Ser Ile Arg

1 5

<210> 100

<211> 5

<212> PRT

<213> Bacillus thuringiensis

<400> 100

Glu Gly Ser Ile Arg

1 5

<210> 101

<211> 9

<212> PRT

<213> Bacillus thuringiensis

<400> 101

Ala Gln Leu Gly Gln Gly Val Tyr Arg

1 5

<210> 102

<211> 6

<212> PRT

<213> Bacillus thuringiensis

<400> 102

Gly Gln Gly Val Tyr Arg

1 5

<210> 103

<211> 6

<212> PRT

<213> Bacillus thuringiensis

<400> 103

Ser Ser Thr Leu Tyr Arg

1 5

<210> 104

<211> 5

<212> PRT

<213> Bacillus thuringiensis

<400> 104

Ser Thr Leu Tyr Arg

1 5

<210> 105

<211> 6

<212> PRT

<213> Bacillus thuringiensis

<400> 105

Ser Val Phe Gly Gln Arg

1 5

<210> 106

<211> 4

<212> PRT

<213> Bacillus thuringiensis

<400> 106

Phe Gly Gln Arg

1

<210> 107

<211> 5

<212> PRT

<213> Bacillus thuringiensis

<400> 107

Ser Gln Leu Thr Arg

1 5

<210> 108

<211> 4

<212> PRT

<213> Bacillus thuringiensis

<400> 108

Gln Leu Thr Arg

1

<210> 109

<211> 6

<212> PRT

<213> Bacillus thuringiensis

<400> 109

Asn Thr Gly Leu Glu Arg

1 5

<210> 110

<211> 7

<212> PRT

<213> Bacillus thuringiensis

<400> 110

Tyr Asn Thr Gly Leu Glu Arg

1 5

<210> 111

<211> 7

<212> PRT

<213> Bacillus thuringiensis

<400> 111

Pro Thr Asn Pro Ala Leu Arg

1 5

<210> 112

<211> 8

<212> PRT

<213> Bacillus thuringiensis

<400> 112

Asp Pro Thr Asn Pro Ala Leu Arg

1 5

<210> 113

<211> 5

<212> PRT

<213> Bacillus thuringiensis

<400> 113

Gly Pro Asp Ser Arg

1 5

<210> 114

<211> 5

<212> PRT

<213> Bacillus thuringiensis

<400> 114

Ser Trp Ile His Arg

1 5

<210> 115

<211> 6

<212> PRT

<213> Bacillus thuringiensis

<400> 115

Ala Thr Ile Asn Ser Arg

1 5

<210> 116

<211> 8

<212> PRT

<213> Bacillus thuringiensis

<400> 116

Asp Ala Ala Thr Ile Asn Ser Arg

1 5

<210> 117

<211> 4

<212> PRT

<213> Bacillus thuringiensis

<400> 117

Ala Ile Ser Arg

1

<210> 118

<211> 4

<212> PRT

<213> Bacillus thuringiensis

<400> 118

Glu Phe Ala Arg

1

<210> 119

<211> 5

<212> PRT

<213> Bacillus thuringiensis

<400> 119

Glu Glu Phe Ala Arg

1 5

<210> 120

<211> 9

<212> PRT

<213> Bacillus thuringiensis

<400> 120

Ser Asn Ser Ser Val Ser Ile Ile Arg

1 5

<210> 121

<211> 7

<212> PRT

<213> Bacillus thuringiensis

<400> 121

Ser Ser Val Ser Ile Ile Arg

1 5

<210> 122

<211> 4

<212> PRT

<213> Bacillus thuringiensis

<400> 122

Ser Met Phe Arg

1

<210> 123

<211> 5

<212> PRT

<213> Bacillus thuringiensis

<400> 123

Val Ser Met Phe Arg

1 5

<210> 124

<211> 8

<212> PRT

<213> Bacillus thuringiensis

<400> 124

Glu Asn Phe Asp Gly Ser Phe Arg

1 5

<210> 125

<211> 4

<212> PRT

<213> Bacillus thuringiensis

<400> 125

Gly Ser Phe Arg

1

<210> 126

<211> 6

<212> PRT

<213> Bacillus thuringiensis

<400> 126

Tyr Ala Glu Ser Phe Arg

1 5

<210> 127

<211> 4

<212> PRT

<213> Bacillus thuringiensis

<400> 127

Asn Gln Phe Arg

1

<210> 128

<211> 4

<212> PRT

<213> Bacillus thuringiensis

<400> 128

Asp Leu Thr Arg

1

<210> 129

<211> 5

<212> PRT

<213> Bacillus thuringiensis

<400> 129

Asn Asp Leu Thr Arg

1 5

<210> 130

<211> 8

<212> PRT

<213> Bacillus thuringiensis

<400> 130

Thr Ile Tyr Thr Asp Ala His Arg

1 5

<210> 131

<211> 6

<212> PRT

<213> Bacillus thuringiensis

<400> 131

Tyr Thr Asp Ala His Arg

1 5

<210> 132

<211> 4

<212> PRT

<213> Bacillus thuringiensis

<400> 132

Pro Leu Thr Lys

1

<210> 133

<211> 8

<212> PRT

<213> Bacillus thuringiensis

<400> 133

Ser Ala Glu Phe Asn Asn Ile Ile

1 5

<210> 134

<211> 4

<212> PRT

<213> Bacillus thuringiensis

<400> 134

Phe Ser His Arg

1

<210> 135

<211> 5

<212> PRT

<213> Bacillus thuringiensis

<400> 135

Gly Phe Ser His Arg

1 5

<210> 136

<211> 7

<212> PRT

<213> Bacillus thuringiensis

<400> 136

Glu Val Leu Gly Gly Glu Arg

1 5

<210> 137

<211> 4

<212> PRT

<213> Bacillus thuringiensis

<400> 137

Gly Gly Glu Arg

1

<210> 138

<211> 7

<212> PRT

<213> Bacillus thuringiensis

<400> 138

Phe Pro Asn Tyr Asp Ser Arg

1 5

<210> 139

<211> 6

<212> PRT

<213> Bacillus thuringiensis

<400> 139

Pro Asn Tyr Asp Ser Arg

1 5

<210> 140

<211> 6

<212> PRT

<213> Bacillus thuringiensis

<400> 140

Pro Ser Ala Val Tyr Arg

1 5

<210> 141

<211> 9

<212> PRT

<213> Bacillus thuringiensis

<400> 141

Ser Gly Thr Val Asp Ser Leu Asp Glu

1 5

<210> 142

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> engineered hybrid toxins

<400> 142

Thr Asp Tyr His Ile Asp Gln Val

1 5

<210> 143

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> engineered hybrid toxins

<400> 143

Asp Tyr His Ile Asp Gln Val

1 5

<210> 144

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> engineered hybrid toxins

<400> 144

Thr Ser Ser Asn Gln Ile Gly Leu Lys

1 5

<210> 145

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> engineered hybrid toxins

<400> 145

Ser Ser Asn Gln Ile Gly Leu Lys

1 5

<210> 146

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> engineered hybrid toxins

<400> 146

Gln Leu Pro Leu Thr Lys

1 5

<210> 147

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> engineered hybrid toxins

<400> 147

Thr Gln Leu Pro Leu Thr Lys

1 5

<210> 148

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> engineered hybrid toxins

<400> 148

Asp Ser Ser Thr Thr Lys

1 5

<210> 149

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> engineered hybrid toxins

<400> 149

Ser Ser Thr Thr Lys

1 5

<210> 150

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> engineered hybrid toxins

<400> 150

Pro Tyr Asp Gly Arg

1 5

<210> 151

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> mutant Cry3A

<400> 151

Gln Leu Pro Leu Val Lys

1 5

<210> 152

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> mutant Cry3A

<400> 152

Thr Gln Leu Pro Leu Val Lys

1 5

<210> 153

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> mutant Cry3A

<400> 153

Ala Leu Asp Ser Ser Thr Thr Lys

1 5

<210> 154

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> mutant Cry3A

<400> 154

Glu Ala Leu Asp Ser Ser Thr Thr Lys

1 5

<210> 155

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> mutant Cry3A

<400> 155

Tyr Ile Asp Lys

1

<210> 156

<211> 7

<212> PRT

<213> Bacillus thuringiensis

<400> 156

Ser Gln Phe Ile Gly Asp Lys

1 5

<210> 157

<211> 5

<212> PRT

<213> Bacillus thuringiensis

<400> 157

Thr Leu Thr Cys Lys

1 5

<210> 158

<211> 7

<212> PRT

<213> Bacillus thuringiensis

<400> 158

Ala Thr Asp Leu Ser Asn Lys

1 5

<210> 159

<211> 8

<212> PRT

<213> Bacillus thuringiensis

<400> 159

Leu Ala Thr Asp Leu Ser Asn Lys

1 5

<210> 160

<211> 9

<212> PRT

<213> Bacillus thuringiensis

<400> 160

Thr Phe Ala Thr Glu Thr Ser Ser Lys

1 5

<210> 161

<211> 8

<212> PRT

<213> Bacillus thuringiensis

<400> 161

Phe Ala Thr Glu Thr Ser Ser Lys

1 5

<210> 162

<211> 4

<212> PRT

<213> Bacillus thuringiensis

<400> 162

Leu Phe Glu Lys

1

<210> 163

<211> 6

<212> PRT

<213> Bacillus thuringiensis

<400> 163

Ser Glu Leu Ile Thr Lys

1 5

<210> 164

<211> 7

<212> PRT

<213> Bacillus thuringiensis

<400> 164

Ala Ser Glu Leu Thr Ile Lys

1 5

<210> 165

<211> 7

<212> PRT

<213> Bacillus thuringiensis

<400> 165

Ser Glu Met Phe Thr Thr Lys

1 5

<210> 166

<211> 7

<212> PRT

<213> Bacillus thuringiensis

<400> 166

Met Asn Glu His Leu Asn Lys

1 5

<210> 167

<211> 7

<212> PRT

<213> Bacillus thuringiensis

<400> 167

Met Asn Glu His Leu Asn Lys

1 5

<210> 168

<211> 4

<212> PRT

<213> Bacillus thuringiensis

<400> 168

Asp Phe Thr Lys

1

<210> 169

<211> 7

<212> PRT

<213> Bacillus thuringiensis

<400> 169

Thr Leu Asp Glu Ile Leu Lys

1 5

<210> 170

<211> 8

<212> PRT

<213> Bacillus thuringiensis

<400> 170

Leu Thr Leu Asp Glu Ile Leu Lys

1 5

<210> 171

<211> 6

<212> PRT

<213> Bacillus thuringiensis

<400> 171

Met Asn Met Ile Phe Lys

1 5

<210> 172

<211> 5

<212> PRT

<213> Bacillus thuringiensis

<400> 172

Asn Met Ile Phe Lys

1 5

<210> 173

<211> 4

<212> PRT

<213> Bacillus thuringiensis

<400> 173

Tyr Val His Lys

1

<210> 174

<211> 4

<212> PRT

<213> Bacillus thuringiensis

<400> 174

Val Asn Ile Leu

1

<210> 175

<211> 8

<212> PRT

<213> Bacillus thuringiensis

<400> 175

Ser Met Leu Ser Asp Val Ile Lys

1 5

<210> 176

<211> 9

<212> PRT

<213> Bacillus thuringiensis

<400> 176

Thr Ser Met Leu Ser Asp Val Ile Lys

1 5

<210> 177

<211> 7

<212> PRT

<213> Bacillus thuringiensis

<400> 177

Asp Ser Phe Ser Thr Tyr Arg

1 5

<210> 178

<211> 8

<212> PRT

<213> Bacillus thuringiensis

<400> 178

Leu Asp Ser Phe Ser Thr Tyr Arg

1 5

<210> 179

<211> 7

<212> PRT

<213> Bacillus thuringiensis

<400> 179

Ile Tyr Gly Asp Met Asp Lys

1 5

<210> 180

<211> 8

<212> PRT

<213> Bacillus thuringiensis

<400> 180

Val Ile Tyr Gly Asp Met Asp Lys

1 5

<210> 181

<211> 9

<212> PRT

<213> Bacillus thuringiensis

<400> 181

Ser Asn Asp Ser Ile Thr Val Leu Lys

1 5

<210> 182

<211> 7

<212> PRT

<213> Bacillus thuringiensis

<400> 182

Ser Gly Asp Ala Asn Val Arg

1 5

<210> 183

<211> 9

<212> PRT

<213> Bacillus thuringiensis

<400> 183

Ser Val Ser Gly Asp Ala Asn Val Arg

1 5

<210> 184

<211> 8

<212> PRT

<213> Bacillus thuringiensis

<400> 184

Leu Leu Asn Asp Ile Ser Gly Lys

1 5

<210> 185

<211> 7

<212> PRT

<213> Bacillus thuringiensis

<400> 185

Leu Asn Asp Ile Ser Gly Lys

1 5

<210> 186

<211> 7

<212> PRT

<213> Bacillus thuringiensis

<400> 186

Ser Ser Glu Ala Glu Tyr Arg

1 5

<210> 187

<211> 8

<212> PRT

<213> Bacillus thuringiensis

<400> 187

Glu Ser Ser Glu Ala Glu Tyr Arg

1 5

<210> 188

<211> 4

<212> PRT

<213> Bacillus thuringiensis

<400> 188

Ser Gly Ala Lys

1

<210> 189

<211> 5

<212> PRT

<213> Bacillus thuringiensis

<400> 189

Met Ser Gly Ala Lys

1 5

<210> 190

<211> 8

<212> PRT

<213> Bacillus thuringiensis

<400> 190

Thr Glu Leu Thr Glu Leu Ala Lys

1 5

<210> 191

<211> 7

<212> PRT

<213> Bacillus thuringiensis

<400> 191

Asp Gly Ser Pro Ala Asp Ile

1 5

<210> 192

<211> 4

<212> PRT

<213> Bacillus thuringiensis

<400> 192

Tyr Glu Ala Lys

1

<210> 193

<211> 5

<212> PRT

<213> Bacillus thuringiensis

<400> 193

Asn Thr Met Leu Arg

1 5

<210> 194

<211> 7

<212> PRT

<213> Bacillus thuringiensis

<400> 194

Ala Ile Asn Thr Met Leu Arg

1 5

<210> 195

<211> 6

<212> PRT

<213> Bacillus thuringiensis

<400> 195

Pro Ser Ile His Leu Lys

1 5

<210> 196

<211> 7

<212> PRT

<213> Bacillus thuringiensis

<400> 196

Asp Tyr Gln Thr Ile Asn Lys

1 5

<210> 197

<211> 4

<212> PRT

<213> Bacillus thuringiensis

<400> 197

Asp Asn Phe Tyr

1

<210> 198

<211> 8

<212> PRT

<213> Bacillus thuringiensis

<400> 198

Pro Asn Glu Tyr Val Ile Thr Lys

1 5

<210> 199

<211> 5

<212> PRT

<213> Bacillus thuringiensis

<400> 199

Ser Pro Ser Glu Lys

1 5

<210> 200

<211> 8

<212> PRT

<213> Bacillus thuringiensis

<400> 200

Leu Glu Ile Ser Pro Ser Glu Lys

1 5

<210> 201

<211> 11

<212> PRT

<213> Bacillus thuringiensis

<400> 201

Asp His Thr Gly Gly Val Asn Ser Gly Thr Lys

1 5 10

<210> 202

<211> 9

<212> PRT

<213> Bacillus thuringiensis

<400> 202

Gly Asn Leu Asn Thr Glu Leu Ser Lys

1 5

<210> 203

<211> 6

<212> PRT

<213> Bacillus thuringiensis

<400> 203

Asn Thr Glu Leu Ser Lys

1 5

<210> 204

<211> 7

<212> PRT

<213> Bacillus thuringiensis

<400> 204

Leu Asn Asp Val Asn Asn Lys

1 5

<210> 205

<211> 6

<212> PRT

<213> Bacillus thuringiensis

<400> 205

Asn Asp Val Asn Asn Lys

1 5

<210> 206

<211> 4

<212> PRT

<213> Bacillus thuringiensis

<400> 206

Asp Leu Asn Lys

1

<210> 207

<211> 7

<212> PRT

<213> Bacillus thuringiensis

<400> 207

Gln Ile Glu Tyr Leu Ser Lys

1 5

<210> 208

<211> 8

<212> PRT

<213> Bacillus thuringiensis

<400> 208

Leu Gln Ile Glu Tyr Leu Ser Lys

1 5

<210> 209

<211> 6

<212> PRT

<213> Bacillus thuringiensis

<400> 209

Gln Glu Ile Ser Asp Lys

1 5

<210> 210

<211> 5

<212> PRT

<213> Bacillus thuringiensis

<400> 210

Tyr Gln Gly Gly Arg

1 5

<210> 211

<211> 7

<212> PRT

<213> Bacillus thuringiensis

<400> 211

Thr Leu Tyr Gln Gly Gly Arg

1 5

<210> 212

<211> 4

<212> PRT

<213> Bacillus thuringiensis

<400> 212

Val Asn Glu Lys

1

<210> 213

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> mutant EPSPS

<400> 213

Glu Ile Val Leu Gln Pro Ile Lys

1 5

<210> 214

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> mutant EPSPS

<400> 214

Pro Val Glu Asp Ala Lys

1 5

<210> 215

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> mutant EPSPS

<400> 215

Val Glu Asp Ala Lys

1 5

<210> 216

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> mutant EPSPS

<400> 216

Ser Gly Thr Val Lys

1 5

<210> 217

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> mutant EPSPS

<400> 217

Gly Thr Val Lys

1

<210> 218

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> mutant EPSPS

<400> 218

Ile Leu Leu Leu Ala Ala

1 5

<210> 219

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> mutant EPSPS

<400> 219

His Tyr Met Leu Gly Ala Leu Arg

1 5

<210> 220

<211> 6

<212> PRT

<213> Streptomyces viridochromogenes

<400> 220

Tyr Thr His Leu Leu Lys

1 5

<210> 221

<211> 5

<212> PRT

<213> Streptomyces viridochromogenes

<400> 221

Thr His Leu Leu Lys

1 5

<210> 222

<211> 4

<212> PRT

<213> Streptomyces viridochromogenes

<400> 222

Ser Pro Glu Arg

1

<210> 223

<211> 5

<212> PRT

<213> Streptomyces viridochromogenes

<400> 223

Gly Phe Trp Gln Arg

1 5

<210> 224

<211> 6

<212> PRT

<213> Streptomyces viridochromogenes

<400> 224

Val Gly Phe Trp Gln Arg

1 5

<210> 225

<211> 8

<212> PRT

<213> Streptomyces viridochromogenes

<400> 225

Ser Thr Val Tyr Val Ser His Arg

1 5

<210> 226

<211> 5

<212> PRT

<213> Streptomyces viridochromogenes

<400> 226

Thr Glu Pro Gln Thr

1 5

<210> 227

<211> 4

<212> PRT

<213> Streptomyces viridochromogenes

<400> 227

Asp Leu Glu Arg

1

<210> 228

<211> 4

<212> PRT

<213> Streptomyces viridochromogenes

<400> 228

Ala Gly Tyr Lys

1

<210> 229

<211> 4

<212> PRT

<213> Streptomyces viridochromogenes

<400> 229

Gly Pro Trp Lys

1

<210> 230

<211> 9

<212> PRT

<213> Streptomyces viridochromogenes

<400> 230

Gly Ile Ala Tyr Ala Gly Pro Trp Lys

1 5

<210> 231

<211> 7

<212> PRT

<213> Streptomyces viridochromogenes

<400> 231

Thr Ser Thr Val Asn Phe Arg

1 5

<210> 232

<211> 7

<212> PRT

<213> Escherichia coli

<400> 232

Pro Met Asp Ala Ala Glu Arg

1 5

<210> 233

<211> 9

<212> PRT

<213> Escherichia coli

<400> 233

Gly Ile Pro Met Asp Ala Ala Glu Arg

1 5

<210> 234

<211> 4

<212> PRT

<213> Escherichia coli

<400> 234

Ala Ile Leu Lys

1

<210> 235

<211> 6

<212> PRT

<213> Escherichia coli

<400> 235

Pro Trp Gln Thr Ile Arg

1 5

<210> 236

<211> 8

<212> PRT

<213> Escherichia coli

<400> 236

Gly Glu Pro Trp Gln Thr Ile Arg

1 5

<210> 237

<211> 9

<212> PRT

<213> Escherichia coli

<400> 237

Ala Asn Glu Ser Pro Val Thr Val Lys

1 5

<210> 238

<211> 5

<212> PRT

<213> Escherichia coli

<400> 238

Pro Val Thr Val Lys

1 5

<210> 239

<211> 6

<212> PRT

<213> Escherichia coli

<400> 239

Leu Thr Gln Pro Val Lys

1 5

<210> 240

<211> 6

<212> PRT

<213> Escherichia coli

<400> 240

Gly Glu Ala Val Ala Lys

1 5

<210> 241

<211> 7

<212> PRT

<213> Escherichia coli

<400> 241

Leu Gly Glu Ala Val Ala Lys

1 5

<210> 242

<211> 8

<212> PRT

<213> Escherichia coli

<400> 242

Gln Asn Tyr Ala Trp Gly Ser Lys

1 5

<210> 243

<211> 7

<212> PRT

<213> Escherichia coli

<400> 243

Asn Tyr Ala Trp Gly Ser Lys

1 5

<210> 244

<211> 8

<212> PRT

<213> Escherichia coli

<400> 244

Asn Ser Glu Ile Gly Phe Ala Lys

1 5

<210> 245

<211> 6

<212> PRT

<213> Escherichia coli

<400> 245

Val Leu Cys Ala Ala Gln

1 5

<210> 246

<211> 7

<212> PRT

<213> Escherichia coli

<400> 246

Trp Met Gly Ala His Pro Lys

1 5

<210> 247

<211> 5

<212> PRT

<213> Escherichia coli

<400> 247

Thr Ala Leu Thr Glu

1 5

<210> 248

<211> 7

<212> PRT

<213> Escherichia coli

<400> 248

Asn Met Gln Gly Glu Glu Lys

1 5

<210> 249

<211> 8

<212> PRT

<213> Escherichia coli

<400> 249

Leu Asn Met Gln Gly Glu Glu Lys

1 5

<210> 250

<211> 8

<212> PRT

<213> Escherichia coli

<400> 250

Ser Leu His Asp Leu Ser Asp Lys

1 5

<210> 251

<211> 6

<212> PRT

<213> Escherichia coli

<400> 251

His Asp Leu Ser Asp Lys

1 5

<210> 252

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> mutant Cry3A

<400> 252

Leu Gln Ser Gly Ala Ser Val Val Ala Gly Pro Arg

1 5 10

<210> 253

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> modified Cry3A peptide

<400> 253

Ser Gly Ala Ser Val Val Ala Gly Pro Arg

1 5 10

<210> 254

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> modified Cry3A peptide

<400> 254

Ser Val Val Ala Gly Pro Arg

1 5

<210> 255

<211> 8

<212> PRT

<213> Bacillus thuringiensis

<400> 255

Phe Thr Thr Gly Thr Asp Leu Lys

1 5

<210> 256

<211> 6

<212> PRT

<213> Bacillus thuringiensis

<400> 256

Thr Gly Thr Asp Leu Lys

1 5

<210> 257

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> mutant EPSPS

<400> 257

Ser Leu Thr Ala Ala Val Thr Ala Ala Gly Gly Asn Ala Thr Tyr Val

1 5 10 15

Leu Asp Gly Val Pro Arg

20

<210> 258

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> dmEPSPS transition 2

<400> 258

Gly Val Pro Arg

1

<210> 259

<211> 615

<212> PRT

<213> Bacillus thuringiensis

<400> 259

Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu

1 5 10 15

Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly

20 25 30

Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser

35 40 45

Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile

50 55 60

Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile

65 70 75 80

Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala

85 90 95

Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu

100 105 110

Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu

115 120 125

Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala

130 135 140

Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val

145 150 155 160

Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser

165 170 175

Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg

180 185 190

Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp His Ala Val

195 200 205

Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg

210 215 220

Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val

225 230 235 240

Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Tyr Pro

245 250 255

Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val

260 265 270

Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu

275 280 285

Gly Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr

290 295 300

Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser Gly His Gln

305 310 315 320

Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro

325 330 335

Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala

340 345 350

Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg

355 360 365

Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp

370 375 380

Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val

385 390 395 400

Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln

405 410 415

Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His

420 425 430

Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile

435 440 445

Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn

450 455 460

Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro Leu Thr Lys Ser Thr

465 470 475 480

Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro Gly Phe Thr Gly

485 490 495

Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gln Ile Ser Thr Leu Arg

500 505 510

Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Tyr Arg Val Arg Ile Arg

515 520 525

Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Thr Ser Ile Asp Gly Arg

530 535 540

Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Met Ser Ser Gly Ser Asn

545 550 555 560

Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe Thr Thr Pro Phe Asn

565 570 575

Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser Ala His Val Phe Asn

580 585 590

Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu Phe Val Pro Ala Glu

595 600 605

Val Thr Phe Glu Ala Glu Tyr

610 615

<210> 260

<211> 653

<212> PRT

<213> Artificial sequence

<220>

<223> engineered hybrid toxins

<400> 260

Met Thr Ser Asn Gly Arg Gln Cys Ala Gly Ile Arg Pro Tyr Asp Gly

1 5 10 15

Arg Gln Gln His Arg Gly Leu Asp Ser Ser Thr Thr Lys Asp Val Ile

20 25 30

Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val Val Gly Phe

35 40 45

Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe Leu Asn Thr

50 55 60

Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu Gln Val Glu

65 70 75 80

Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn Lys Ala Leu

85 90 95

Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr Val Ser Ala

100 105 110

Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg Asn Pro His

115 120 125

Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His Phe

130 135 140

Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu Val Leu Phe

145 150 155 160

Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe Leu Leu Lys

165 170 175

Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys Glu Asp Ile

180 185 190

Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu Tyr Thr Asp

195 200 205

His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu Arg Gly Ser

210 215 220

Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg Glu Met Thr

225 230 235 240

Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr Asp Val Arg

245 250 255

Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp Val Leu Thr

260 265 270

Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly Thr Thr Phe

275 280 285

Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe Asp Tyr Leu

290 295 300

His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr Tyr Gly Asn

305 310 315 320

Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr Arg Pro Ser

325 330 335

Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly Asn Lys Ser

340 345 350

Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys Val Tyr Arg

355 360 365

Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala Val Tyr Ser

370 375 380

Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln Thr Asp Glu

385 390 395 400

Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly Ala Val Ser

405 410 415

Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro Leu

420 425 430

Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys Phe Leu Met

435 440 445

Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr His Lys Ser

450 455 460

Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr Gln Leu Pro

465 470 475 480

Leu Thr Lys Ser Thr Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly

485 490 495

Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gln

500 505 510

Ile Ser Thr Leu Arg Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Tyr

515 520 525

Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Thr

530 535 540

Ser Ile Asp Gly Arg Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Met

545 550 555 560

Ser Ser Gly Ser Asn Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe

565 570 575

Thr Thr Pro Phe Asn Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser

580 585 590

Ala His Val Phe Asn Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu

595 600 605

Phe Val Pro Ala Glu Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg

610 615 620

Ala Gln Lys Ala Val Asn Glu Leu Phe Thr Ser Ser Asn Gln Ile Gly

625 630 635 640

Leu Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln Val

645 650

<210> 261

<211> 598

<212> PRT

<213> Artificial sequence

<220>

<223> mutant Cry3A

<400> 261

Met Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys

1 5 10 15

Asp Val Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Val

20 25 30

Val Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr Asn Phe

35 40 45

Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe Met Glu

50 55 60

Gln Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala Lys Asn

65 70 75 80

Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu Asp Tyr

85 90 95

Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Ala Ala Pro Phe Arg

100 105 110

Asn Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu

115 120 125

Ser His Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr Glu

130 135 140

Val Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Phe

145 150 155 160

Leu Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu Lys

165 170 175

Glu Asp Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln Glu

180 185 190

Tyr Thr Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys Leu

195 200 205

Arg Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg Arg

210 215 220

Glu Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu Tyr

225 230 235 240

Asp Val Arg Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg Asp

245 250 255

Val Leu Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr Gly

260 265 270

Thr Thr Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu Phe

275 280 285

Asp Tyr Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly Tyr

290 295 300

Tyr Gly Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser Thr

305 310 315 320

Arg Pro Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr Gly

325 330 335

Asn Lys Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu Lys

340 345 350

Val Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser Ala

355 360 365

Val Tyr Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp Gln

370 375 380

Thr Asp Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val Gly

385 390 395 400

Ala Val Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp

405 410 415

Glu Pro Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met Cys

420 425 430

Phe Leu Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp Thr

435 440 445

His Lys Ser Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile Thr

450 455 460

Gln Leu Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser Val

465 470 475 480

Val Ala Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr Glu

485 490 495

Asn Gly Ser Ala Ala Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr Ser

500 505 510

Gln Lys Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile Thr

515 520 525

Phe Thr Leu Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Tyr Phe Asp

530 535 540

Lys Thr Ile Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe Asn Leu

545 550 555 560

Ala Ser Phe Ser Thr Pro Phe Glu Leu Ser Gly Asn Asn Leu Gln Ile

565 570 575

Gly Val Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr Ile Asp Lys Ile

580 585 590

Glu Phe Ile Pro Val Asn

595

<210> 262

<211> 789

<212> PRT

<213> Artificial sequence

<220>

<223> modified Vip3A

<400> 262

Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro Ser Phe

1 5 10 15

Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp

20 25 30

Ile Met Asn Met Ile Phe Lys Thr Asp Thr Gly Gly Asp Leu Thr Leu

35 40 45

Asp Glu Ile Leu Lys Asn Gln Gln Leu Leu Asn Asp Ile Ser Gly Lys

50 55 60

Leu Asp Gly Val Asn Gly Ser Leu Asn Asp Leu Ile Ala Gln Gly Asn

65 70 75 80

Leu Asn Thr Glu Leu Ser Lys Glu Ile Leu Lys Ile Ala Asn Glu Gln

85 90 95

Asn Gln Val Leu Asn Asp Val Asn Asn Lys Leu Asp Ala Ile Asn Thr

100 105 110

Met Leu Arg Val Tyr Leu Pro Lys Ile Thr Ser Met Leu Ser Asp Val

115 120 125

Ile Lys Gln Asn Tyr Ala Leu Ser Leu Gln Ile Glu Tyr Leu Ser Lys

130 135 140

Gln Leu Gln Glu Ile Ser Asp Lys Leu Asp Ile Ile Asn Val Asn Val

145 150 155 160

Leu Ile Asn Ser Thr Leu Thr Glu Ile Thr Pro Ala Tyr Gln Arg Ile

165 170 175

Lys Tyr Val Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr Glu Thr

180 185 190

Ser Ser Lys Val Lys Lys Asp Gly Ser Pro Ala Asp Ile Leu Asp Glu

195 200 205

Leu Thr Glu Leu Thr Glu Leu Ala Lys Ser Val Thr Lys Asn Asp Val

210 215 220

Asp Gly Phe Glu Phe Tyr Leu Asn Thr Phe His Asp Val Met Val Gly

225 230 235 240

Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr Ala Ser Glu Leu Ile

245 250 255

Thr Lys Glu Asn Val Lys Thr Ser Gly Ser Glu Val Gly Asn Val Tyr

260 265 270

Asn Phe Leu Ile Val Leu Thr Ala Leu Gln Ala Gln Ala Phe Leu Thr

275 280 285

Leu Thr Thr Cys Arg Lys Leu Leu Gly Leu Ala Asp Ile Asp Tyr Thr

290 295 300

Ser Ile Met Asn Glu His Leu Asn Lys Glu Lys Glu Glu Phe Arg Val

305 310 315 320

Asn Ile Leu Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro Asn Tyr Ala

325 330 335

Lys Val Lys Gly Ser Asp Glu Asp Ala Lys Met Ile Val Glu Ala Lys

340 345 350

Pro Gly His Ala Leu Ile Gly Phe Glu Ile Ser Asn Asp Ser Ile Thr

355 360 365

Val Leu Lys Val Tyr Glu Ala Lys Leu Lys Gln Asn Tyr Gln Val Asp

370 375 380

Lys Asp Ser Leu Ser Glu Val Ile Tyr Gly Asp Met Asp Lys Leu Leu

385 390 395 400

Cys Pro Asp Gln Ser Glu Gln Ile Tyr Tyr Thr Asn Asn Ile Val Phe

405 410 415

Pro Asn Glu Tyr Val Ile Thr Lys Ile Asp Phe Thr Lys Lys Met Lys

420 425 430

Thr Leu Arg Tyr Glu Val Thr Ala Asn Phe Tyr Asp Ser Ser Thr Gly

435 440 445

Glu Ile Asp Leu Asn Lys Lys Lys Val Glu Ser Ser Glu Ala Glu Tyr

450 455 460

Arg Thr Leu Ser Ala Asn Asp Asp Gly Val Tyr Met Pro Leu Gly Val

465 470 475 480

Ile Ser Glu Thr Phe Leu Thr Pro Ile Asn Gly Phe Gly Leu Gln Ala

485 490 495

Asp Glu Asn Ser Arg Leu Ile Thr Leu Thr Cys Lys Ser Tyr Leu Arg

500 505 510

Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys Glu Thr Lys Leu Ile

515 520 525

Val Pro Pro Ser Gly Phe Ile Ser Asn Ile Val Glu Asn Gly Ser Ile

530 535 540

Glu Glu Asp Asn Leu Glu Pro Trp Lys Ala Asn Asn Lys Asn Ala Tyr

545 550 555 560

Val Asp His Thr Gly Gly Val Asn Gly Thr Lys Ala Leu Tyr Val His

565 570 575

Lys Asp Gly Gly Ile Ser Gln Phe Ile Gly Asp Lys Leu Lys Pro Lys

580 585 590

Thr Glu Tyr Val Ile Gln Tyr Thr Val Lys Gly Lys Pro Ser Ile His

595 600 605

Leu Lys Asp Glu Asn Thr Gly Tyr Ile His Tyr Glu Asp Thr Asn Asn

610 615 620

Asn Leu Glu Asp Tyr Gln Thr Ile Asn Lys Arg Phe Thr Thr Gly Thr

625 630 635 640

Asp Leu Lys Gly Val Tyr Leu Ile Leu Lys Ser Gln Asn Gly Asp Glu

645 650 655

Ala Trp Gly Asp Asn Phe Ile Ile Leu Glu Ile Ser Pro Ser Glu Lys

660 665 670

Leu Leu Ser Pro Glu Leu Ile Asn Thr Asn Asn Trp Thr Ser Thr Gly

675 680 685

Ser Thr Asn Ile Ser Gly Asn Thr Leu Thr Leu Tyr Gln Gly Gly Arg

690 695 700

Gly Ile Leu Lys Gln Asn Leu Gln Leu Asp Ser Phe Ser Thr Tyr Arg

705 710 715 720

Val Tyr Phe Ser Val Ser Gly Asp Ala Asn Val Arg Ile Arg Asn Ser

725 730 735

Arg Glu Val Leu Phe Glu Lys Arg Tyr Met Ser Gly Ala Lys Asp Val

740 745 750

Ser Glu Met Phe Thr Thr Lys Phe Glu Lys Asp Asn Phe Tyr Ile Glu

755 760 765

Leu Ser Gln Gly Asn Asn Leu Tyr Gly Gly Pro Ile Val His Phe Tyr

770 775 780

Asp Val Ser Ile Lys

785

<210> 263

<211> 445

<212> PRT

<213> Artificial sequence

<220>

<223> double mutant 5-enolpyruvylshikimate-3-phosphate synthase

<400> 263

Met Ala Gly Ala Glu Glu Ile Val Leu Gln Pro Ile Lys Glu Ile Ser

1 5 10 15

Gly Thr Val Lys Leu Pro Gly Ser Lys Ser Leu Ser Asn Arg Ile Leu

20 25 30

Leu Leu Ala Ala Leu Ser Glu Gly Thr Thr Val Val Asp Asn Leu Leu

35 40 45

Asn Ser Glu Asp Val His Tyr Met Leu Gly Ala Leu Arg Thr Leu Gly

50 55 60

Leu Ser Val Glu Ala Asp Lys Ala Ala Lys Arg Ala Val Val Val Gly

65 70 75 80

Cys Gly Gly Lys Phe Pro Val Glu Asp Ala Lys Glu Glu Val Gln Leu

85 90 95

Phe Leu Gly Asn Ala Gly Ile Ala Met Arg Ser Leu Thr Ala Ala Val

100 105 110

Thr Ala Ala Gly Gly Asn Ala Thr Tyr Val Leu Asp Gly Val Pro Arg

115 120 125

Met Arg Glu Arg Pro Ile Gly Asp Leu Val Val Gly Leu Lys Gln Leu

130 135 140

Gly Ala Asp Val Asp Cys Phe Leu Gly Thr Asp Cys Pro Pro Val Arg

145 150 155 160

Val Asn Gly Ile Gly Gly Leu Pro Gly Gly Lys Val Lys Leu Ser Gly

165 170 175

Ser Ile Ser Ser Gln Tyr Leu Ser Ala Leu Leu Met Ala Ala Pro Leu

180 185 190

Ala Leu Gly Asp Val Glu Ile Glu Ile Ile Asp Lys Leu Ile Ser Ile

195 200 205

Pro Tyr Val Glu Met Thr Leu Arg Leu Met Glu Arg Phe Gly Val Lys

210 215 220

Ala Glu His Ser Asp Ser Trp Asp Arg Phe Tyr Ile Lys Gly Gly Gln

225 230 235 240

Lys Tyr Lys Ser Pro Lys Asn Ala Tyr Val Glu Gly Asp Ala Ser Ser

245 250 255

Ala Ser Tyr Phe Leu Ala Gly Ala Ala Ile Thr Gly Gly Thr Val Thr

260 265 270

Val Glu Gly Cys Gly Thr Thr Ser Leu Gln Gly Asp Val Lys Phe Ala

275 280 285

Glu Val Leu Glu Met Met Gly Ala Lys Val Thr Trp Thr Glu Thr Ser

290 295 300

Val Thr Val Thr Gly Pro Pro Arg Glu Pro Phe Gly Arg Lys His Leu

305 310 315 320

Lys Ala Ile Asp Val Asn Met Asn Lys Met Pro Asp Val Ala Met Thr

325 330 335

Leu Ala Val Val Ala Leu Phe Ala Asp Gly Pro Thr Ala Ile Arg Asp

340 345 350

Val Ala Ser Trp Arg Val Lys Glu Thr Glu Arg Met Val Ala Ile Arg

355 360 365

Thr Glu Leu Thr Lys Leu Gly Ala Ser Val Glu Glu Gly Pro Asp Tyr

370 375 380

Cys Ile Ile Thr Pro Pro Glu Lys Leu Asn Val Thr Ala Ile Asp Thr

385 390 395 400

Tyr Asp Asp His Arg Met Ala Met Ala Phe Ser Leu Ala Ala Cys Ala

405 410 415

Glu Val Pro Val Thr Ile Arg Asp Pro Gly Cys Thr Arg Lys Thr Phe

420 425 430

Pro Asp Tyr Phe Asp Val Leu Ser Thr Phe Val Lys Asn

435 440 445

<210> 264

<211> 191

<212> PRT

<213> Streptomyces viridochromogenes

<400> 264

Met Ser Pro Glu Arg Arg Pro Val Glu Ile Arg Pro Ala Thr Ala Ala

1 5 10 15

Asp Met Ala Ala Val Cys Asp Ile Val Asn His Tyr Ile Glu Thr Ser

20 25 30

Thr Val Asn Phe Arg Thr Glu Pro Gln Thr Pro Gln Glu Trp Ile Asp

35 40 45

Asp Leu Glu Arg Leu Gln Asp Arg Tyr Pro Trp Leu Val Ala Glu Val

50 55 60

Glu Gly Val Val Ala Gly Ile Ala Tyr Ala Gly Pro Trp Lys Ala Arg

65 70 75 80

Asn Ala Tyr Asp Trp Thr Val Glu Ser Thr Val Tyr Val Ser His Arg

85 90 95

His Gln Arg Leu Gly Leu Gly Ser Thr Leu Tyr Thr His Leu Leu Lys

100 105 110

Ser Met Glu Ala Gln Gly Phe Lys Ser Val Val Ala Val Ile Gly Leu

115 120 125

Pro Asn Asp Pro Ser Val Arg Leu His Glu Ala Leu Gly Tyr Thr Ala

130 135 140

Arg Gly Thr Leu Arg Ala Ala Gly Tyr Lys His Gly Gly Trp His Asp

145 150 155 160

Val Gly Phe Trp Gln Arg Asp Phe Glu Leu Pro Ala Pro Pro Arg Pro

165 170 175

Val Arg Pro Val Thr Gln Ile Pro Asp Arg Ser Asn Ile Trp Gln

180 185 190

<210> 265

<211> 391

<212> PRT

<213> Escherichia coli

<400> 265

Met Gln Lys Leu Ile Asn Ser Val Gln Asn Tyr Ala Trp Gly Ser Lys

1 5 10 15

Thr Ala Leu Thr Glu Leu Tyr Gly Met Glu Asn Pro Ser Ser Gln Pro

20 25 30

Met Ala Glu Leu Trp Met Gly Ala His Pro Lys Ser Ser Ser Arg Val

35 40 45

Gln Asn Ala Ala Gly Asp Ile Val Ser Leu Arg Asp Val Ile Glu Ser

50 55 60

Asp Lys Ser Thr Leu Leu Gly Glu Ala Val Ala Lys Arg Phe Gly Glu

65 70 75 80

Leu Pro Phe Leu Phe Lys Val Leu Cys Ala Ala Gln Pro Leu Ser Ile

85 90 95

Gln Val His Pro Asn Lys His Asn Ser Glu Ile Gly Phe Ala Lys Glu

100 105 110

Asn Ala Ala Gly Ile Pro Met Asp Ala Ala Glu Arg Asn Tyr Lys Asp

115 120 125

Pro Asn His Lys Pro Glu Leu Val Phe Ala Leu Thr Pro Phe Leu Ala

130 135 140

Met Asn Ala Phe Arg Glu Phe Ser Glu Ile Val Ser Leu Leu Gln Pro

145 150 155 160

Val Ala Gly Ala His Pro Ala Ile Ala His Phe Leu Gln Gln Pro Asp

165 170 175

Ala Glu Arg Leu Ser Glu Leu Phe Ala Ser Leu Leu Asn Met Gln Gly

180 185 190

Glu Glu Lys Ser Arg Ala Leu Ala Ile Leu Lys Ser Ala Leu Asp Ser

195 200 205

Gln Gln Gly Glu Pro Trp Gln Thr Ile Arg Leu Ile Ser Glu Phe Tyr

210 215 220

Pro Glu Asp Ser Gly Leu Phe Ser Pro Leu Leu Leu Asn Val Val Lys

225 230 235 240

Leu Asn Pro Gly Glu Ala Met Phe Leu Phe Ala Glu Thr Pro His Ala

245 250 255

Tyr Leu Gln Gly Val Ala Leu Glu Val Met Ala Asn Ser Asp Asn Val

260 265 270

Leu Arg Ala Gly Leu Thr Pro Lys Tyr Ile Asp Ile Pro Glu Leu Val

275 280 285

Ala Asn Val Lys Phe Glu Ala Lys Pro Ala Asn Gln Leu Leu Thr Gln

290 295 300

Pro Val Lys Gln Gly Ala Glu Leu Asp Phe Pro Ile Pro Val Asp Asp

305 310 315 320

Phe Ala Phe Ser Leu His Asp Leu Ser Asp Lys Glu Thr Thr Ile Ser

325 330 335

Gln Gln Ser Ala Ala Ile Leu Phe Cys Val Glu Gly Asp Ala Thr Leu

340 345 350

Trp Lys Gly Ser Gln Gln Leu Gln Leu Lys Pro Gly Glu Ser Ala Phe

355 360 365

Ile Ala Ala Asn Glu Ser Pro Val Thr Val Lys Gly His Gly Arg Leu

370 375 380

Ala Arg Val Tyr Asn Lys Leu

385 390

<210> 266

<211> 391

<212> PRT

<213> Artificial sequence

<220>

<223> MIR604 PMI

<400> 266

Met Gln Lys Leu Ile Asn Ser Val Gln Asn Tyr Ala Trp Gly Ser Lys

1 5 10 15

Thr Ala Leu Thr Glu Leu Tyr Gly Met Glu Asn Pro Ser Ser Gln Pro

20 25 30

Met Ala Glu Leu Trp Met Gly Ala His Pro Lys Ser Ser Ser Arg Val

35 40 45

Gln Asn Ala Ala Gly Asp Ile Val Ser Leu Arg Asp Ala Ile Glu Ser

50 55 60

Asp Lys Ser Thr Leu Leu Gly Glu Ala Val Ala Lys Arg Phe Gly Glu

65 70 75 80

Leu Pro Phe Leu Phe Lys Val Leu Cys Ala Ala Gln Pro Leu Ser Ile

85 90 95

Gln Val His Pro Asn Lys His Asn Ser Glu Ile Gly Phe Ala Lys Glu

100 105 110

Asn Ala Ala Gly Ile Pro Met Asp Ala Ala Glu Arg Asn Tyr Lys Asp

115 120 125

Pro Asn His Lys Pro Glu Leu Val Phe Ala Leu Thr Pro Phe Leu Ala

130 135 140

Met Asn Ala Phe Arg Glu Phe Ser Glu Ile Val Ser Leu Leu Gln Pro

145 150 155 160

Val Ala Gly Ala His Pro Ala Ile Ala His Phe Leu Gln Gln Pro Asp

165 170 175

Ala Glu Arg Leu Ser Glu Leu Phe Ala Ser Leu Leu Asn Met Gln Gly

180 185 190

Glu Glu Lys Ser Arg Ala Leu Ala Ile Leu Lys Ser Ala Leu Asp Ser

195 200 205

Gln His Gly Glu Pro Trp Gln Thr Ile Arg Leu Ile Ser Glu Phe Tyr

210 215 220

Pro Glu Asp Ser Gly Leu Phe Ser Pro Leu Leu Leu Asn Val Val Lys

225 230 235 240

Leu Asn Pro Gly Glu Ala Met Phe Leu Phe Ala Glu Thr Pro His Ala

245 250 255

Tyr Leu Gln Gly Val Ala Leu Glu Val Met Ala Asn Ser Asp Asn Val

260 265 270

Leu Arg Ala Gly Leu Thr Pro Lys Tyr Ile Asp Ile Pro Glu Leu Val

275 280 285

Ala Asn Val Lys Phe Glu Ala Lys Pro Ala Asn Gln Leu Leu Thr Gln

290 295 300

Pro Val Lys Gln Gly Ala Glu Leu Asp Phe Pro Ile Pro Val Asp Asp

305 310 315 320

Phe Ala Phe Ser Leu His Asp Leu Ser Asp Lys Glu Thr Thr Ile Ser

325 330 335

Gln Gln Ser Ala Ala Ile Leu Phe Cys Val Glu Gly Asp Ala Thr Leu

340 345 350

Trp Lys Gly Ser Gln Gln Leu Gln Leu Lys Pro Gly Glu Ser Ala Phe

355 360 365

Ile Ala Ala Asn Glu Ser Pro Val Thr Val Lys Gly His Gly Arg Leu

370 375 380

Ala Arg Val Tyr Asn Lys Leu

385 390

<210> 267

<211> 881

<212> PRT

<213> Artificial sequence

<220>

<223> engineered hybrid toxins

<400> 267

Met Asp Ile Leu Asn Ser Ile Thr Ile Tyr Thr Asp Ala His Arg Gly

1 5 10 15

Glu Tyr Tyr Trp Ser Gly His Gln Ile Met Ala Ser Pro Val Gly Phe

20 25 30

Ser Gly Pro Glu Phe Thr Phe Pro Leu Tyr Gly Thr Met Gly Asn Ala

35 40 45

Ala Pro Gln Gln Arg Ile Val Ala Gln Leu Gly Gln Gly Val Tyr Arg

50 55 60

Thr Leu Ser Ser Thr Leu Tyr Arg Arg Pro Phe Asn Ile Gly Ile Asn

65 70 75 80

Asn Gln Gln Leu Ser Val Leu Asp Gly Thr Glu Phe Ala Tyr Gly Thr

85 90 95

Ser Ser Asn Leu Pro Ser Ala Val Tyr Arg Lys Ser Gly Thr Val Asp

100 105 110

Ser Leu Asp Glu Ile Pro Pro Gln Asn Asn Asn Val Pro Pro Arg Gln

115 120 125

Gly Phe Ser His Arg Leu Ser His Val Ser Met Phe Arg Ser Gly Phe

130 135 140

Ser Asn Ser Ser Val Ser Ile Ile Arg Ala Pro Met Phe Ser Trp Ile

145 150 155 160

His Arg Ser Ala Glu Phe Asn Asn Ile Ile Ala Ser Asp Ser Ile Thr

165 170 175

Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val

180 185 190

Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser

195 200 205

Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu Pro

210 215 220

Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg

225 230 235 240

Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn

245 250 255

Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr

260 265 270

Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser Phe

275 280 285

Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile Asp

290 295 300

Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Leu Glu Ala Glu Tyr Asn

305 310 315 320

Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Thr Asn

325 330 335

Gln Leu Gly Leu Lys Thr Asn Val Thr Asp Tyr His Ile Asp Gln Val

340 345 350

Ser Asn Leu Val Thr Tyr Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys

355 360 365

Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu

370 375 380

Arg Asn Leu Leu Gln Asp Ser Asn Phe Lys Asp Ile Asn Arg Gln Pro

385 390 395 400

Glu Arg Gly Trp Gly Gly Ser Thr Gly Ile Thr Ile Gln Gly Gly Asp

405 410 415

Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Ser Gly Thr Phe Asp Glu

420 425 430

Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys

435 440 445

Ala Phe Thr Arg Tyr Gln Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp

450 455 460

Leu Glu Ile Tyr Ser Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn

465 470 475 480

Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile

485 490 495

Gly Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp Asn

500 505 510

Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His

515 520 525

Ser His His Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn

530 535 540

Glu Asp Leu Gly Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly

545 550 555 560

His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val

565 570 575

Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp

580 585 590

Lys Arg Glu Lys Leu Glu Trp Glu Thr Asn Ile Val Tyr Lys Glu Ala

595 600 605

Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Gln Leu

610 615 620

Gln Ala Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val

625 630 635 640

His Ser Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly

645 650 655

Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala

660 665 670

Phe Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn

675 680 685

Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu Glu

690 695 700

Gln Asn Asn Gln Arg Ser Val Leu Val Val Pro Glu Trp Glu Ala Glu

705 710 715 720

Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg

725 730 735

Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His

740 745 750

Glu Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu

755 760 765

Glu Glu Ile Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Val

770 775 780

Asn Gln Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr

785 790 795 800

Asn Glu Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu

805 810 815

Lys Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn Arg

820 825 830

Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr Val Thr Lys Glu

835 840 845

Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu

850 855 860

Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu

865 870 875 880

Glu

<210> 268

<211> 605

<212> PRT

<213> Bacillus thuringiensis

<400> 268

Met Glu Asn Asn Ile Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn

1 5 10 15

Asn Pro Glu Val Glu Ile Leu Asn Glu Glu Arg Ser Thr Gly Arg Leu

20 25 30

Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe

35 40 45

Val Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly

50 55 60

Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gln Ile Glu Gln

65 70 75 80

Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile Thr

85 90 95

Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu Ile Tyr Ile Glu Ala Leu

100 105 110

Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gln Leu Arg Glu Asp Val

115 120 125

Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Leu Ile Thr Ala Ile Asn

130 135 140

Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr Val

145 150 155 160

Gln Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe

165 170 175

Gly Gln Gly Trp Gly Leu Asp Ile Ala Thr Val Asn Asn His Tyr Asn

180 185 190

Arg Leu Ile Asn Leu Ile His Arg Tyr Thr Lys His Cys Leu Asp Thr

195 200 205

Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gln Trp

210 215 220

Ala Arg Phe Asn Gln Phe Arg Arg Asp Leu Thr Leu Thr Val Leu Asp

225 230 235 240

Ile Val Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro Ile Gln

245 250 255

Thr Ser Ser Gln Leu Thr Arg Glu Ile Tyr Thr Ser Ser Val Ile Glu

260 265 270

Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gly Phe Asn Arg Ala Glu

275 280 285

Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe

290 295 300

Val Thr Ala Glu Thr Val Arg Ser Gln Thr Val Trp Gly Gly His Leu

305 310 315 320

Val Ser Ser Arg Asn Thr Ala Gly Asn Arg Ile Asn Phe Pro Ser Tyr

325 330 335

Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp Pro

340 345 350

Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly

355 360 365

Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gln

370 375 380

Gln Thr Gly Thr Asn His Thr Arg Thr Phe Arg Asn Ser Gly Thr Ile

385 390 395 400

Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro Trp

405 410 415

Asn Asp Tyr Ser His Val Leu Asn His Val Thr Phe Val Arg Trp Pro

420 425 430

Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe Ser Trp

435 440 445

Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile

450 455 460

Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr

465 470 475 480

Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr

485 490 495

Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu

500 505 510

Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu

515 520 525

Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe

530 535 540

Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser

545 550 555 560

Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser

565 570 575

Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile

580 585 590

Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Leu Glu

595 600 605

<210> 269

<211> 123

<212> PRT

<213> Bacillus thuringiensis

<400> 269

Met Ser Ala Arg Glu Val His Ile Asp Val Asn Asn Lys Thr Gly His

1 5 10 15

Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp Gly Gly Arg Trp Arg

20 25 30

Thr Ser Pro Thr Asn Val Ala Asn Asp Gln Ile Lys Thr Phe Val Ala

35 40 45

Glu Ser Asn Gly Phe Met Thr Gly Thr Glu Gly Thr Ile Tyr Tyr Ser

50 55 60

Ile Asn Gly Glu Ala Glu Ile Ser Leu Tyr Phe Asp Asn Pro Phe Ala

65 70 75 80

Gly Ser Asn Lys Tyr Asp Gly His Ser Asn Lys Ser Gln Tyr Glu Ile

85 90 95

Ile Thr Gln Gly Gly Ser Gly Asn Gln Ser His Val Thr Tyr Thr Ile

100 105 110

Gln Thr Thr Ser Ser Arg Tyr Gly His Lys Ser

115 120

<210> 270

<211> 354

<212> PRT

<213> Bacillus thuringiensis

<400> 270

Met Leu Asp Thr Asn Lys Val Tyr Glu Ile Ser Asn His Ala Asn Gly

1 5 10 15

Leu Tyr Ala Ala Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu

20 25 30

Met Asn Lys Asn Asp Asp Asp Ile Asp Asp Tyr Asn Leu Lys Trp Phe

35 40 45

Leu Phe Pro Ile Asp Asp Asp Gln Tyr Ile Ile Thr Ser Tyr Ala Ala

50 55 60

Asn Asn Cys Lys Val Trp Asn Val Asn Asn Asp Lys Ile Asn Val Ser

65 70 75 80

Thr Tyr Ser Ser Thr Asn Ser Ile Gln Lys Trp Gln Ile Lys Ala Asn

85 90 95

Gly Ser Ser Tyr Val Ile Gln Ser Asp Asn Gly Lys Val Leu Thr Ala

100 105 110

Gly Thr Gly Gln Ala Leu Gly Leu Ile Arg Leu Thr Asp Glu Ser Ser

115 120 125

Asn Asn Pro Asn Gln Gln Trp Asn Leu Thr Ser Val Gln Thr Ile Gln

130 135 140

Leu Pro Gln Lys Pro Ile Ile Asp Thr Lys Leu Lys Asp Tyr Pro Lys

145 150 155 160

Tyr Ser Pro Thr Gly Asn Ile Asp Asn Gly Thr Ser Pro Gln Leu Met

165 170 175

Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn Asp Pro Asn Ile Asp

180 185 190

Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Leu Lys Lys Tyr

195 200 205

Gln Tyr Trp Gln Arg Ala Val Gly Ser Asn Val Ala Leu Arg Pro His

210 215 220

Glu Lys Lys Ser Tyr Thr Tyr Glu Trp Gly Thr Glu Ile Asp Gln Lys

225 230 235 240

Thr Thr Ile Ile Asn Thr Leu Gly Phe Gln Ile Asn Ile Asp Ser Gly

245 250 255

Met Lys Phe Asp Ile Pro Glu Val Gly Gly Gly Thr Asp Glu Ile Lys

260 265 270

Thr Gln Leu Asn Glu Glu Leu Lys Ile Glu Tyr Ser His Glu Thr Lys

275 280 285

Ile Met Glu Lys Tyr Gln Glu Gln Ser Glu Ile Asp Asn Pro Thr Asp

290 295 300

Gln Ser Met Asn Ser Ile Gly Phe Leu Thr Ile Thr Ser Leu Glu Leu

305 310 315 320

Tyr Arg Tyr Asn Gly Ser Glu Ile Arg Ile Met Gln Ile Gln Thr Ser

325 330 335

Asp Asn Asp Thr Tyr Asn Val Thr Ser Tyr Pro Asn His Gln Gln Ala

340 345 350

Leu Leu