阅读说明：本技术 一种整合蛋白质组和糖蛋白组的定量分析方法 (Quantitative analysis method for integrating proteome and glycoprotein ) 是由 张勇 赵婉君 杨浩 程惊秋 于 2019-08-16 设计创作，主要内容包括：本发明提供了一种整合蛋白质组和糖蛋白质组的定量分析方法,包括以下步骤：(1)蛋白质加热变性后,进行还原烷基化处理；(2)在超滤管中使用胰蛋白酶将蛋白质酶切成肽段,离心即得肽段；(3)亲水作用色谱HILIC填料结合肽段,洗涤除杂,再洗脱得完整N-糖肽；(4)加入三氟乙酸并加热去除N-糖链外围唾液酸,得到去唾液酸的N-糖肽；(5)使用HCD-MS/MS分析肽段得到蛋白质组数据,并使用MaxQuant定性定量分析；(6)使用SCE-HCD-MS/MS分析完整N-糖肽和去唾液酸的N-糖肽,使用Xcalibur软件定性定量分析。本发明能简单、快速有效地实现蛋白质组和糖蛋白质组的定量分析,在疾病的发生发展机制研究以及新型生物标志物发现等方面具有良好的应用前景。(The invention provides a quantitative analysis method for integrating proteome and glycoproteome, which comprises the following steps: (1) carrying out reductive alkylation treatment after protein heating denaturation; (2) using trypsin to cut the protein enzyme into peptide segments in an ultrafiltration tube, and centrifuging to obtain the peptide segments; (3) combining the hydrophilic interaction chromatography HILIC filler with the peptide segment, washing to remove impurities, and then washing to obtain complete N-glycopeptide; (4) adding trifluoroacetic acid and heating to remove sialic acid on the periphery of the N-sugar chain to obtain N-glycopeptide with sialic acid removed; (5) analyzing the peptide fragment by using HCD-MS/MS to obtain proteome data, and performing qualitative and quantitative analysis by using MaxQuant; (6) the intact N-glycopeptides and desialidated N-glycopeptides were analyzed using SCE-HCD-MS/MS and qualitatively and quantitatively using the Xcalibur software. The invention can simply, quickly and effectively realize the quantitative analysis of proteome and glycoproteome, and has good application prospect in the aspects of disease occurrence and development mechanism research, novel biomarker discovery and the like.)

1. A method for the quantitative analysis of integrated proteomes and glycoproteomes, comprising the steps of:

(1) carrying out reduction alkylation treatment after heating denaturation of the protein, opening a disulfide bond of the protein and carrying out alkylation closure;

(2) using trypsin to cut the protein enzyme into peptide segments in an ultrafiltration tube, and centrifuging to obtain the peptide segments;

(3) combining the hydrophilic interaction chromatography HILIC filler with the peptide segment, washing to remove impurities, and eluting to obtain complete N-glycopeptide;

(4) adding trifluoroacetic acid and heating to remove sialic acid on the periphery of the N-sugar chain to obtain N-glycopeptide with sialic acid removed;

(5) analyzing the peptide fragment by using HCD-MS/MS to obtain proteome data, and performing qualitative and quantitative analysis by using MaxQuant software;

(6) the complete N-glycopeptide and the desialidated N-glycopeptide were analyzed by SCE-HCD-MS/MS to obtain glycoproteome data, and qualitative and quantitative analysis was performed by using Xcalibur software.

2. The integrated analysis method of claim 1, wherein the reductive alkylation in step (1) is performed by using dithiothreitol with a final concentration of 10-40 mmol/L at 50-60 ℃ for 45 minutes with shaking, adding iodoacetamide with a final concentration of 25-100 mmol/L, and performing a light-shielding reaction at room temperature for 0.5-2 hours.

3. The integrated assay of claim 1, wherein the filtration pore size of the ultrafiltration tube of step (2) is 30kDa or 10 kDa.

4. The integrated assay of claim 1, wherein the cleavage reaction of step (2) is performed according to the following parameters:

enzyme dosage: 2-10 mug of enzyme per 100 mug protein;

reaction solution: 50mmol/L ammonium bicarbonate;

reaction temperature: 37 ℃;

reaction time: 2-16 h.

5. The integrated analytical method of claim 1, wherein the HILIC filler of step (3) is Venusil HILIC filler of Boraijeger, and has a particle size of 3 μm, 5 μm and/or 10 μm and a pore size of 3 μm, 5 μm and/or 10 μm

6. The integrated assay of claim 1, wherein the binding conditions of step (3) are spin binding for 2h at room temperature. The mobile phase used for washing and impurity removal is a mixed solution of water, acetonitrile and trifluoroacetic acid, and the volume ratio is as follows in sequence: (10-30): (70-90): 0.2. when eluting, 0.1-1% (v/v) trifluoroacetic acid solution is used for elution.

7. The integrated assay of claim 1, wherein the preferred reaction solution in step (4) is 0.1-1% (v) trifluoroacetic acid solution, the reaction temperature is 80 ℃ and the reaction time is 1-2 h.

8. The integrated assay of claim 1, wherein the chromatographic conditions of steps (5) and (6) are:

a chromatographic column: the Magic C18 column had an inner diameter of 75 μm, a length of 15em, and a C18 particle size of 3 μm.

Mobile phase A liquid: 0.1% (v) aqueous trifluoroacetic acid;

mobile phase B liquid: 0.1% (v) trifluoroacetic acid in 80% (v) acetonitrile;

linear gradient of mobile phase B: 5-32%;

flow rate of mobile phase: 0.3. mu.L/min.

9. The analytical method of claim 1, wherein the mass spectrometry conditions of step (5) are:

the fragmentation pattern of the mass spectrometer was HCD, the time was 78min and the flow rate was 0.3. mu.L/min. The mass range of the first-stage mass spectrum ion scanning is 300-1400 Da, the resolution is 120000, and the AGC is 5e⁵Maximum IT is 50ms, exclusion time is 18 s; setting the ion window of the secondary mass spectrum to be 1.6m/z, setting the detection mode to be an ion trap and setting AGC to be 5e³The maximum IT is 35 ms.

10. The analytical method of claim 1, wherein the mass spectrometry conditions of step (6) are:

the fragmentation pattern of the mass spectrometer was HCD, the time was 78min and the flow rate was 0.3. mu.L/min. The mass range of the primary mass spectrum ion scanning is 800-2000 Da, the resolution is 120000, and the AGC is 2e⁵Maximum IT is 100ms, and exclusion time is 15 s; setting the ion window of the secondary mass spectrum to be 2m/z, setting the detection mode to be an orbit trap,AGC is 5e⁵Maximum IT is 250ms, step fragmentation energy is turned on, HCD energy is 30% +/-10%.

11. The analysis method according to claim 1, wherein the data processing method of step (6) is:

a. manually accumulating MS1 spectrograms to generate a mass spectrogram after deconvolution;

b. normalizing the charge state to a single charge, and removing other isotope peaks except the single isotope peak to obtain a peptide fragment signal;

c. generating a signal list, and calculating the quality of the peptide fragment according to the Y1 value;

d. subtracting the mass of the peptide fragment from the mass of the monoisotopic peak to obtain a candidate glycoform; the deconvolved peak intensities correspond to the abundance of the aforementioned glycoforms.

Technical Field

The invention relates to the field of biochemistry, and particularly relates to a quantitative analysis method for an integrated proteome and a glycoproteome.

Background

It has been found that a large number of patients with diseases have abnormal changes in their important proteins and their glycosylation modifications, including the type of protein, the amount of protein expressed, the structure of sugar chains, the site of glycosylation modification, the degree of glycosylation modification, and the like. Therefore, the important proteins and the glycosylation modification changes thereof are deeply understood, a new method for analyzing the proteome and the glycoproteome simultaneously is developed, and the acquisition of multidimensional information is helpful for understanding the occurrence and development mechanism of diseases and providing diagnostic markers and therapeutic targets.

However, quantitative analysis methods for integrating proteome and glycoproteome are lacking because of many difficulties and challenges in technical approaches.

Firstly, in the pretreatment of a sample for separately studying proteome, because the glycosylation modification abundance is very low, the signal of glycopeptide is inhibited by non-glycopeptide in mass spectrometry detection, and thus, only qualitative and quantitative analysis can be carried out on the protein.

Secondly, in the pretreatment of a sample for independently researching a glycoproteome, the abundance of the glycosylated peptide segment is very low, so that an effective glycopeptide enrichment method is needed for enriching glycopeptides and removing the interference of non-glycopeptides, and only the information of the glycopeptides can be obtained in mass spectrum detection.

Finally, conventional collision-based mass spectrometry fragmentation methods such as collision fragmentation (CID) preferentially fragment sugar chains, resulting in very few peptide backbone fragments and very low oxonium ions, are difficult to identify for intact glycopeptides, and therefore require the search for alternative fragmentation modes such as mass spectrometry for step energy HCD fragmentation (SCE-HCD-MS/MS).

Furthermore, the prior art studies of proteomes and glycoproteins have been mainly separate studies, and there have been few reports of integrated quantitative analysis of proteomes and glycoproteins (intact N-glycopeptides and asialo N-glycopeptides). Zhang Hui et al established an analytical method for integrating proteomes and glycoproteomes to study prostate cancer cell lines, but the proteome quantification method was iTRAQ marker quantification and the glycoproteomes were deglycosylated glycopeptides, so this method was costly, long-lived, and lost glycosylation site-specific information.

Disclosure of Invention

The object of the present invention is to establish a method for integrating the quantitative determination of a marker-free proteome and the quantitative analysis of an intact N-glycopeptide and a asialo N-glycopeptide.

The technical scheme of the invention comprises the following steps:

a method for the quantitative analysis of integrated proteomes and glycoproteomes comprising the steps of:

(1) carrying out reduction alkylation treatment after heating denaturation of the protein, opening a disulfide bond of the protein and carrying out alkylation closure;

(2) using trypsin to cut the protein enzyme into peptide segments in an ultrafiltration tube, and centrifuging to obtain the peptide segments;

(3) combining the hydrophilic interaction chromatography HILIC filler with the peptide segment, washing to remove impurities, and eluting to obtain complete N-glycopeptide;

(4) adding trifluoroacetic acid and heating to remove sialic acid on the periphery of the N-sugar chain to obtain N-glycopeptide with sialic acid removed;

(5) analyzing the peptide fragment by using HCD-MS/MS to obtain proteome data, and performing qualitative and quantitative analysis by using MaxQuant software;

(6) the complete N-glycopeptide and the desialidated N-glycopeptide were analyzed by SCE-HCD-MS/MS to obtain glycoproteome data, and qualitative and quantitative analysis was performed by using Xcalibur software.

In the method, dithiothreitol with a final concentration of 10-40 mmol/L is used for the reductive alkylation treatment in the step (1) and is subjected to oscillation reaction at 50-60 ℃ for 45 minutes, and iodoacetamide with a final concentration of 25-100 mmol/L is added for reaction at room temperature in a dark place for 0.5-2 hours.

As the method, the filtration pore size of the ultrafiltration tube in the step (2) is 30kDa or 10 kDa.

The method is as described above, and the enzyme digestion reaction in the step (2) follows the following parameters:

enzyme dosage: 2-10 mug of enzyme per 100 mug protein;

reaction solution: 50mmol/L ammonium bicarbonate;

reaction temperature: 37 ℃;

reaction time: 2-16 h.

As in the previous method, the HILIC filler in the step (3) is Venusil HILIC filler of Boraijeger, the particle diameter is 3 mu m, 5 mu m and/or 10 mu m, and the pore diameter is

As the method, the binding condition in step (3) is spin-binding at room temperature for 2 h. The mobile phase used for washing and impurity removal is a mixed solution of water, acetonitrile and trifluoroacetic acid, and the volume ratio is as follows in sequence: (10-30): (70-90): 0.2. When eluting, 0.1-1% (v/v) trifluoroacetic acid solution is used for elution.

As mentioned above, the preferable reaction solution in step (4) is 0.1-1% (v) trifluoroacetic acid solution, the reaction temperature is 80 ℃, and the reaction time is 1-2 h.

The method, the chromatographic conditions of the step (5) and the step (6) are as follows:

a chromatographic column: the Magic C18 column had an inner diameter of 75 μm, a length of 15em, and a C18 particle size of 3 μm.

Mobile phase A liquid: 0.1% (v) aqueous trifluoroacetic acid;

mobile phase B liquid: 0.1% (v) trifluoroacetic acid in 80% (v) acetonitrile;

linear gradient of mobile phase B: 5-32%;

flow rate of mobile phase: 0.3. mu.L/min.

The method as described above, wherein the mass spectrometry conditions in step (5) are as follows:

mass spectrometryThe fragmentation pattern was HCD, the time was 78min and the flow rate was 0.3. mu.L/min. The mass range of the first-stage mass spectrum ion scanning is 300-1400 Da, the resolution is 120000, and the AGC is 5e⁵Maximum IT is 50ms, exclusion time is 18 s; setting the ion window of the secondary mass spectrum to be 1.6m/z, setting the detection mode to be an ion trap and setting AGC to be 5e³The maximum IT is 35 ms.

The method as described above, wherein the mass spectrometry conditions in step (6) are as follows:

the fragmentation pattern of the mass spectrometer was HCD, the time was 78min and the flow rate was 0.3. mu.L/min. The mass range of the primary mass spectrum ion scanning is 800-2000 Da, the resolution is 120000, and the AGC is 2e⁵Maximum IT is 100ms, and exclusion time is 15 s; setting the ion window of the secondary mass spectrum to be 2m/z, setting the detection mode to be an orbital trap and setting AGC to be 5e⁵Maximum IT is 250ms, step fragmentation energy is turned on, HCD energy is 30% +/-10%.

As the foregoing method, the data processing method in step (6) is:

a. manually accumulating MS1 spectrograms to generate a mass spectrogram after deconvolution;

b. normalizing the charge state to a single charge, and removing other isotope peaks except the single isotope peak to obtain a peptide fragment signal;

c. generating a signal list, and calculating the quality of the peptide fragment according to the Y1 value;

d. subtracting the mass of the peptide fragment from the mass of the monoisotopic peak to obtain a candidate glycoform; the deconvolved peak intensities correspond to the abundance of the aforementioned glycoforms.

The invention has the following beneficial effects:

firstly, the invention uses ultrafiltration method to quickly obtain protein glycopeptide, then obtains complete N-glycopeptide by hydrophilic interaction chromatography enrichment, and finally removes sialic acid quickly by acid method to obtain N-glycopeptide without sialic acid. Compared with the prior method (quantitative research on proteome by using an ultrafiltration tube assisted enzyme cutting method, or quantitative research on deglycosylated peptide fragments after removing N-sugar chains by adopting PNGase F enzyme, or qualitative research on the complete N-glycopeptide obtained by enrichment, which takes about 24 hours in total), the method has the advantages of simple steps, time saving (about 12 hours in total), easy operation and low cost, and can simultaneously obtain qualitative and quantitative information of proteome and glycoprotein (complete N-glycopeptide, desialylated N-glycopeptide).

Secondly, the mass spectrometer used for the analysis of the invention is Orbitrap Fusion Lumos, and the HCD-MS/MS mode is adopted for the peptide fragment, and the SCE-HCD-MS/MS mode is adopted for the complete N-glycopeptide and the asialo N-glycopeptide. The peptide fragment framework fragments are relatively complete, and the N-glycopeptide also contains rich sugar fragment information and Y ion (Y ion formed by the peptide fragment framework and the sugar chain fragments) information, thereby being beneficial to accurate qualitative and quantitative analysis in the later period.

Thirdly, the invention uses Xcalibur software to realize the relative quantification of the complete N-glycopeptide of the glycoprotein and the N-glycopeptide with sialic acid removed, thereby providing a new method for the quantification of the complete N-glycopeptide, reducing the complexity of the complete glycopeptide by the sialic acid removal treatment, avoiding the influence of the sialic acid and ensuring more reliable quantification result.

The method has low cost and short time, can simultaneously obtain the three-dimensional information of proteome, complete N-glycopeptide and asialo N-glycopeptide, and has good application prospect in the aspects of novel biomarker discovery, disease occurrence and development mechanism research and the like.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The foregoing aspects of the present invention are explained in further detail below with reference to specific embodiments. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is a flow chart of the quantitative analysis of integrated proteomes and glycoproteomes.

FIG. 2 is a peptide fragment first order graph of IgG.

FIG. 3 is a primary spectrum of the complete N-glycopeptide of IgG.

FIG. 4 is a first order spectrum of asialo N-glycopeptide of IgG.

FIG. 5 shows the proteomic quantification results of IgG.

FIG. 6 is a graph showing the results of quantitative analysis of IgG glycoproteome; in the molecules of the Glycan (polysaccharide) column in the figure, blue squares represent glucose, green circles represent mannose, red diamonds represent sialic acid, and yellow circles represent galactose; # represents a glycosylation modification site; before stands for intact glycopeptide; after stands for asialoglycopeptide.

Detailed Description

The experimental procedure of the present invention is shown in FIG. 1.

In order to make the technical solutions of the present invention better understood, the present invention is further described in detail below with reference to the accompanying drawings.

Abbreviations: IAM, iodoacetamide; TFA, trifluoroacetic acid; ACN, acetonitrile; ABC, ammonium bicarbonate.