阅读说明：本技术 苯乙酮衍生物应用于不饱和脂质分析的方法 (Method for applying acetophenone derivatives to unsaturated lipid analysis ) 是由 瑕瑜 赵婧 于 2020-04-01 设计创作，主要内容包括：本发明公开了一种结合光化学衍生和串联质谱分析对脂质双键位置进行结构解析的方法。脂质脂肪链上的双键在254纳米紫外光激发下会与苯乙酮衍生物2’,4’,6’-三氟苯乙酮(triFAP)或4’-三氟甲基苯乙酮(FMAP)发生高效的Paternò-Büchì(PB)反应,形成一对含四元环结构的异构体产物。在串联质谱中,该对异构体产物能产生一对质荷比固定的诊断离子,从而用于碳碳双键位置的确定。对于同时含有多个碳碳双键位置异构体的脂质,利用诊断离子的强度比例,可以直接得出各异构体的相对含量。该方法可与鸟枪法或液相色谱分离相结合,实现对复杂样品中的脂质高通量、高灵敏、精细结构解析。(The invention discloses a method for carrying out structural analysis on a lipid double bond position by combining photochemical derivatization and tandem mass spectrometry, wherein double bonds on a lipid aliphatic chain can be efficiently subjected to a Patern oa-B ü ch (PB) reaction with an acetophenone derivative 2 ', 4', 6 '-trifluorophenylacetophenone (triFAP) or 4' -trifluoromethylacetophenone (FMAP) under the excitation of 254nm ultraviolet light to form a pair of isomer products containing a quaternary ring structure.)

1. An unsaturated lipid analysis method, comprising:

(1) preparing a lipid sample solution containing 2 ', 4', 6 '-trifluoro acetophenone or 4' -trifluoro methylacetophenone;

(2) under the action of ultraviolet light, leading a lipid sample and 2 ', 4', 6 '-trifluoro acetophenone or 4' -trifluoromethyl acetophenone to generate derivatization reaction to obtain a 2 ', 4', 6 '-trifluoro acetophenone derivatization product or a 4' -trifluoromethyl acetophenone derivatization product;

(3) performing mass spectrometry on the 2 ', 4', 6 '-trifluoro acetophenone derivatization product or the 4' -trifluoro methylacetophenone derivatization product, and judging the position of the carbon-carbon double bond in the lipid through the diagnostic ion generated in the mass spectrometry.

2. The unsaturated lipid analysis method according to claim 1, wherein the lipid sample is an unsaturated lipid standard or a complex biological sample containing unsaturated lipids.

3. The unsaturated lipid analysis method according to claim 1, wherein the wavelength of the ultraviolet light is 254 nm.

4. The method for analyzing unsaturated lipids according to claim 1, wherein the derivatization reaction is performed for 2 to 25 seconds.

5. The method for analyzing unsaturated lipids according to claim 1, wherein the collision energy used in the mass spectrometry is 35-45 eV.

6. The unsaturated lipid analysis method according to claim 1, wherein the instrument used in the mass spectrometry is a triple quadrupole mass spectrometer or a beam-type CID mass spectrometer.

7. The method of claim 1, wherein the mass spectrometry is performed by shotgun analysis, direct injection, or combined liquid chromatography-mass spectrometry.

8. The unsaturated lipid analysis method according to claim 1, further comprising:

and establishing a relative quantitative curve of the carbon-carbon double bond isomers according to the total intensity ratio of the diagnostic ions and the content ratio of the carbon-carbon double bond isomers in the lipid sample.

9. Use of the unsaturated lipid analysis method of any one of claims 1 to 8 for analyzing bovine liver extract.

Technical Field

The invention relates to the technical field of mass spectrometry, in particular to a method for applying acetophenone derivatives to unsaturated lipid analysis, and more particularly relates to high-flux mass spectrometry detection and analysis on fine structures of the acetophenone derivatives by utilizing the characteristic breaking behavior of products obtained by photoreaction of lipids and the acetophenone derivatives in mass spectrometry.

Background

Lipids are important structural scaffolds of cell membranes, and are involved in a variety of cell signaling and metabolic processes, an important subset of which, unsaturated lipids, characterized by at least one carbon-carbon double bond (C ═ C) distributed on the alkyl chain. Related studies indicate that the distribution of unsaturated lipids exhibits regular changes in relevant organelles involved in the secretory pathway and in some differentiated structures such as neurites; and the C ═ C isomer ratio has been shown to have abnormal changes in breast cancer cells and in body fluids of patients of type T2D, and is a potential disease biomarker. Thus, the identification of unsaturated lipids is of great interest, and the accurate localization of C ═ C in a sensitive and high-throughput manner is a challenge.

Mass spectrometry is a common analytical detection means, and the characteristics of high sensitivity, high selectivity and high throughput make it increasingly interesting for lipidomics application. In recent years, with the development of direct injection "shotgun lipidomics" and separation-based "liquid chromatography-mass spectrometry" methods, the precise structure analysis of lipids and the biological effects caused by the precise structure become the hot spots of research. Although high resolution mass spectrometry is easy to obtain the precise molecular composition of lipid molecules and determine the lipid species, tandem mass spectrometry is still the most intuitive means for characterizing deep structures of lipids. To solve the problem of locating C ═ C, various Mass Spectrometry (MS) methods and chemical derivatization strategies were designed and developed, such as ozone-induced dissociation (OzID), ultraviolet light dissociation (UVPD), m-chloroperoxybenzoic acid (m-CPBA) epoxidation reaction and collision-induced dissociation (CID) -MS/MS, etc. However, their use in lipidomics is not very widespread, limited by instrument modifications and complexity requirements.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. To this end, an object of the present invention is to propose an unsaturated lipid analysis method. The method can be combined with a shotgun method or liquid chromatography separation, and realizes high-flux, high-sensitivity and fine structure analysis of lipid in a complex sample.

The inventors previously developed a C ═ C specific modification method (PB-MS/MS) that can be used in tandem mass spectrometry, which utilizes the Patern oa-B ü ch im reaction between acetone and C ═ C (PB reaction, PB Rxn) to achieve rapid double bond derivatization, is compatible with shotgun and liquid chromatography lipidomics, and has been successfully applied to the analysis of fatty acids, glycerophospholipids and cholesterol esters.

In view of this, in one aspect of the invention, the invention provides a method for unsaturated lipid analysis. According to an embodiment of the present invention, the unsaturated lipid analysis method includes: (1) preparing a lipid sample solution containing 2 ', 4', 6 '-trifluoro acetophenone or 4' -trifluoro methylacetophenone; (2) under the action of ultraviolet light, leading a lipid sample and 2 ', 4', 6 '-trifluoro acetophenone or 4' -trifluoromethyl acetophenone to generate derivatization reaction to obtain a 2 ', 4', 6 '-trifluoro acetophenone derivatization product or a 4' -trifluoromethyl acetophenone derivatization product; (3) performing mass spectrometry on the 2 ', 4', 6 '-trifluoro acetophenone derivatization product or the 4' -trifluoro methylacetophenone derivatization product, and judging the position of the carbon-carbon double bond in the lipid through the diagnostic ion generated in the mass spectrometry.

The inventor finds that 2 ', 4', 6 '-trifluoro acetophenone (called TriFAP for short, molecular weight 174) or 4' -trifluoro methylacetophenone (called FMAP for short, molecular weight 188) can generate derivatization reaction with lipid under the action of ultraviolet light, and the method mainly shows that a new peak of +174Da (TriFAP) or +188Da (FMAP) appears in a primary mass spectrogram after the reaction in a mass spectrum, and the conversion rate is about 35-40%. The serial mass spectrometry is carried out on the detected reactant of the lipid and the acetophenone derivatives, a secondary spectrogram mainly can see a diagnostic ion pair (mass-to-charge ratio difference is 142Da or 156Da), a water loss peak (-18Da) and a neutral loss peak (-174Da or-188 Da) of a reagent except the fragment of the lipid, and the diagnosis of the position of the double bond mainly depends on the diagnostic ion pair. Under the excitation of ultraviolet light, the acetophenone derivatives react with lipid double bonds to generate oxygen-containing four-membered rings, under the action of Collision Induced Dissociation (CID) of mass spectra, the oxygen-containing four-membered rings are cracked along the direction vertical to the synthesis direction to generate a new olefin structure and a new carbonyl structure, and fragment ions with charges on the head part can be observed in a mass spectrogram. Due to different attack directions, two oxygen-containing four-membered rings are generated, a pair of fragment ions can be observed aiming at each double bond, and the mass-to-charge ratios of the pair of fragment ions have a fixed difference, so that the positions of the double bonds can be judged. Therefore, the method can realize high-flux, high-sensitivity and fine structure analysis of the lipid in the complex sample, provides a direction for searching a new biomarker, and provides more comprehensive lipid analysis for the biological diagnosis of a clinical sample in the future.

In addition, the unsaturated lipid analysis method according to the above embodiment of the present invention may further have the following additional technical features:

in some embodiments of the invention, the lipid sample is an unsaturated lipid standard or a complex biological sample containing unsaturated lipids.

In some embodiments of the invention, the ultraviolet light has a wavelength of 254 nm.

In some embodiments of the invention, the derivatization is carried out for a time period of 2 to 25 seconds.

In some embodiments of the invention, the mass spectrometry uses a collision energy of 30-45 eV.

In some embodiments of the invention, the instrument employed in the mass spectrometry is a triple quadrupole mass spectrometer or a beam-type CID mass spectrometer.

In some embodiments of the invention, the mass spectrometry is performed by a shotgun method, a direct injection method, or a combined liquid chromatography-mass spectrometry method.

In some embodiments of the invention, the unsaturated lipid analysis method further comprises: and establishing a relative quantitative curve of the carbon-carbon double bond isomers according to the total intensity ratio of the diagnostic ions and the content ratio of the carbon-carbon double bond isomers in the lipid sample.

In another aspect of the present invention, the present invention proposes the use of the unsaturated lipid analysis method of the above example in the analysis of bovine liver extract. As mentioned above, the unsaturated lipid analysis method provided by the invention can realize high-throughput, high-sensitivity and fine structure analysis of lipids in complex samples. The inventor applies the unsaturated lipid analysis method to the analysis of the bovine liver extract, and 194 unsaturated lipid molecules including 41 pairs of double bond isomers are identified in the bovine liver polar lipid extract through the structure analysis process. Therefore, the unsaturated lipid analysis method realizes high-flux, high-sensitivity and fine structure analysis of the lipid in the complex sample, provides a direction for searching a new biomarker, and provides more comprehensive lipid analysis for biological diagnosis of a clinical sample in the future.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 is a schematic view of an off-line microfluidic photoreactor device for PB reaction according to an embodiment of the invention;

FIG. 2a is a first order mass spectrum of PC16:0/18:1 (5. mu.M) after reaction with 2mM TriFAP; FIG. 2b is a CID spectrum of the reaction product of PC16:0/18:1 (collision energy around 40 eV); FIG. 2c is a chromatogram of a reaction product of PC16:0/18:1 after separation by a reverse phase chromatographic column, wherein the extracted ions are m/z 934.6 in a first-order spectrum; FIG. 2d is a chromatogram of a reaction product of PC16:0/18:1 after separation by a reverse phase chromatographic column, wherein the extracted ions are fragment ions m/z918.6 (B), m/z 760.6(Y), m/z 792.5(R) and m/z 650.5(G) in a secondary spectrum; FIGS. 2e-h are CID spectra corresponding to 14.5-19.5min in the chromatogram;

FIG. 3a is a first order mass spectrum of PC16:0/18:1 (5. mu.M) after reaction with 2mM FMAP; FIG. 3b is a CID spectrum of the reaction product of PC16:0/18:1 (collision energy around 40 eV); FIG. 3c is a chromatogram of a PC16:0/18:1 reaction product after separation by a reverse phase chromatography columnExtracting ions as m/z 948.6 in the first-order spectrum; FIG. 3d is a chromatogram of the reaction product PC16:0/18:1 after separation by a reverse phase chromatographic column, wherein the extracted ion is fragment ion m/z 930.6 (-H) in a secondary spectrum₂O),m/z760.6(-R),m/z 806.5(F_O) And m/z 650.5 (F)_A)；

FIG. 4a is the distribution of fragment ions as a function of the CID collision energy of the photoreaction product of PC16:0/18:1 and TriFAP, and FIG. 4b is the CID spectrum at a collision energy of 30 eV;

FIG. 5a is a CID spectrum of the product of equimolar mixtures of PC 18:1 (. DELTA.9)/18: 1 (. DELTA.9) and PC 18:1 (. DELTA.6)/18: 1 (. DELTA.6) after reaction with triFAP; FIG. 5b is a calibration curve for the total ionic strength and molar ratio of PC isomers for diagnosis;

FIG. 6a is a first order spectrum of a polar lipid extract from bovine liver tissue after photoreaction with acetone; FIG. 6b is a first order spectrum of bovine liver tissue polar lipid extract after photoreaction with triFAP; FIG. 6c is PC distribution information obtained by PIS 184 on an unreacted complex sample; FIG. 6d is a CID spectrum of the acetate ion adduct of unreacted PC 36:1 in anionic mode; FIG. 6e is the CID spectrum of PC 36:1 obtained by TriFAP PB-MS/MS in positive ion mode.

Detailed Description

The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

In one aspect of the invention, the invention provides a method for analyzing unsaturated lipids. According to an embodiment of the present invention, the unsaturated lipid analysis method includes: (1) preparing a lipid sample solution containing 2 ', 4', 6 '-trifluoro acetophenone or 4' -trifluoro methylacetophenone; (2) under the action of ultraviolet light, leading a lipid sample and 2 ', 4', 6 '-trifluoro acetophenone or 4' -trifluoromethyl acetophenone to generate derivatization reaction to obtain a 2 ', 4', 6 '-trifluoro acetophenone derivatization product or a 4' -trifluoromethyl acetophenone derivatization product; (3) performing mass spectrometry on the 2 ', 4', 6 '-trifluoro acetophenone derivatization product or the 4' -trifluoromethyl acetophenone derivatization product, and judging the position of a carbon-carbon double bond in the lipid through a diagnostic ion generated in the mass spectrometry.

According to some embodiments of the invention, the lipid sample may be an unsaturated lipid standard or a complex biological sample containing unsaturated lipids.

According to some embodiments of the invention, the ultraviolet light has a wavelength of 254 nm.

According to some embodiments of the invention, the derivatization is carried out for a time of 2 to 25s, such as 2s, 5s, 10s, 15s, 20s, 25s, and the like. This can further increase the conversion rate of the reaction between the lipid and the acetophenone derivative.

According to some embodiments of the present invention, the mass spectrometry uses a collision energy of 30-45 eV, such as 30eV, 35eV, 40eV, 45eV, and the like. The inventor finds in research that the collision energy is further beneficial to the peak value of the intensity of diagnostic ions in mass spectrometry, and if the collision energy is too high, the derivative product can be broken into other ions, which adversely affects the mass spectrometry. Specifically, fig. 4a shows the distribution of fragment ions as a function of the CID collision energy of the photoreaction product of PC16:0/18:1 and triFAP, and fig. 4b shows the CID spectrum at a collision energy of 30 eV. Under the mass spectrum condition, the detection limit of the method is about 0.5nM, and the method is also suitable for determining the position of the double bond connected with the ether bond.

According to some embodiments of the present invention, the mass spectrometer may be a triple quadrupole mass spectrometer or a beam-type CID mass spectrometer, and the sample introduction method may be a shotgun method, a direct sample introduction method, or a liquid chromatography-mass spectrometry combination method, thereby further facilitating the realization of high-sensitivity and high-flux structural analysis of unsaturated lipids.

According to some embodiments of the invention, the method for analyzing unsaturated lipids further comprises: and establishing a relative quantitative curve of the carbon-carbon double bond isomers according to the total intensity ratio of the diagnostic ions and the content ratio of the carbon-carbon double bond isomers in the lipid sample. FIG. 5a is a CID spectrum of the product of equimolar mixtures of PC 18:1 (. DELTA.9)/18: 1 (. DELTA.9) and PC 18:1 (. DELTA.6)/18: 1 (. DELTA.6) after reaction with triFAP; FIG. 5b is a calibration curve of total diagnostic ionic strength and molar ratio of PC isomers.

The unsaturated lipid analysis method is described in detail below according to a specific example of the present invention. According to an embodiment of the present invention, the unsaturated lipid analysis method includes:

step (1), referring to FIG. 1, the lipid extract was dissolved in acetonitrile: 20mM ammonium acetate aqueous solution at a volume ratio of 1:1, which additionally contained 2% ethanol and 2mM tripfap (or FMAP). The prepared solution is deoxidized for about 5min under nitrogen flow. Then, the solution is subjected to off-line PB reaction for about 20 s. The liquid after the reaction was loaded into a sodium lance or vial.

The offline PB reaction in the step (1) is performed in a microfluidic reaction device. A254 nm low-pressure ultraviolet mercury lamp was placed in parallel with a fused silica capillary (outer diameter: 363. mu.m, inner diameter: 100 μm), at a distance of about 0.5cm, and at an irradiation length of about 5 cm. The fused quartz capillary as microflow reactor is connected via two-way to injector, which is driven by injection pump to regulate the flow rate of sample and further the reaction time.

And (2) analyzing the unreacted lipid solution, if the sodium spraying method is adopted, inserting a stainless steel wire into the liquid of a sodium spray pipe, aligning the tip of the sodium spray pipe with a sample feeding small hole of the mass spectrum, and if the liquid chromatography-mass spectrum combined method is adopted, placing a small bottle containing about 30 mu L of solution in a sample feeding device for further mass spectrum analysis.

The liquid chromatography used in step (2) above was Exion L C AC system (Sciex) equipped with a degasser, two pumps, an autosampler and a column oven, the lipids in the above samples were separated by a hydrophilic interaction column (HI L IC) model 150mM × 2.1.1 mM, silica spheres,2.7 μm (Sigma-Aldrich), column temperature 30 ℃ mobile phase A10 mM ammonium acetate in water, mobile phase B acetonitrile containing 0.2% acetic acid, mobile phase gradient 0-5min 90% to 85% B, 5-15min 85% to 70% B, 15-20min 70% to 70% B, 20-21min 70% to 90% B, 22-25min 90% to 90% B, flow rate of mobile phase 0.2m L/min, and 3 μm L μm each.

The mass spectrometer used in the above step (2) was QTRAP 4500 from Sciex. The mass spectral parameters for neutral loss scan or precursor ion scan in positive ion mode were set as: air curtain air 30 psi; a collision gas high; a voltage of 4500V; the temperature is 400 ℃; spraying 30psi of mist; auxiliary heating gas 30 psi; the declustering voltage is 80 eV; the collision energy was 45 eV. When the chain information is analyzed in the negative ion mode, the collision energy is changed to 40eV, and the remaining parameters are the same as those described above.

And (3) carrying out PB derivatization and CID analysis on the unsaturated lipid detected in the step (2). The TriFAP (or FMAP) reaction product of the lipid molecule, i.e. the molecule relative to the protonated lipid +174Da (or 188Da), is CID in positive ion mode, and the position of the carbon-carbon double bond is determined according to the ion difference of 142Da (or 156Da) in the spectrogram.

Setting the mass spectrum parameters in the step (3) to be 30psi of air curtain air; a collision gas high; a voltage of 4500V; the temperature is 400 ℃; spraying 30psi of mist; auxiliary heating gas 30 psi; the declustering voltage is 80 eV; the collision energy was 40 eV.

In another aspect of the present invention, the present invention proposes the use of the unsaturated lipid analysis method of the above example in the analysis of bovine liver extract. As mentioned above, the unsaturated lipid analysis method provided by the invention can realize high-throughput, high-sensitivity and fine structure analysis of lipids in complex samples. The inventor applies the unsaturated lipid analysis method to the analysis of the bovine liver extract, and 194 unsaturated lipid molecules including 41 pairs of double bond isomers are identified in the bovine liver polar lipid extract through the structure analysis process. Therefore, the unsaturated lipid analysis method realizes high-flux, high-sensitivity and fine structure analysis of the lipid in the complex sample, provides a direction for searching a new biomarker, and provides more comprehensive lipid analysis for biological diagnosis of a clinical sample in the future.

The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.