A complete N-glycopeptide quantitative analysis method and system

Abstract

The present invention belongs to the field of proteomics technology, and specifically relates to a method and system for quantitative analysis of complete N-glycopeptides. The method of the present invention comprises the following steps: step 1, performing glycoprotein sample pre-treatment, and performing mass spectrometry detection to obtain N-glycopeptide mass spectrometry data; step 2, using library search software to perform complete N-glycopeptide qualitative analysis; step 3, using PANDA software to perform complete N-glycopeptide quantitative analysis. The parameter setting of PANDA software is optimized based on the characteristics of N-glycopeptide mass spectrometry data. The present invention further provides a system for implementing the above method (named N-glycoQuant). The present invention proposes a complete N-glycopeptide mass spectrometry data quantitative solution for the quantitative analysis of complete N-glycopeptides, which can be used for large-scale clinical sample complete N-glycopeptide quantitative analysis, discover new markers, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of proteomics, and in particular relates to a method and system for quantitative analysis of intact N-glycopeptides. Background Art

[0002] Glycoproteomics, a key branch of proteomics, studies the composition and dynamics of glycoproteins in cells, tissues, or organisms, focusing on the glycoproteome. Previous studies have shown that protein glycosylation is widely involved in various physiological and pathological regulation, and abnormal changes in glycoproteins or glycosylation modifications are often closely associated with the development and progression of various diseases. At the glycoprotein level, over one-third of FDA-approved tumor markers are glycoproteins, such as the breast cancer marker Her2 / neu, the liver cancer marker AFP, the pancreatic cancer marker CA19-9, the prostate cancer marker PSA, and the ovarian cancer markers OVA1 and CA125. At the glycoform and glycosylation site levels, it has also been found that a large number of glycans and glycosylation modification sites undergo abnormal changes during disease development and progression. Therefore, glycoproteomics research currently focuses on analyzing intact glycopeptides, which can simultaneously obtain three-dimensional information about glycoproteins, glycomodification sites, and glycans. This is of great significance for disease diagnosis, classification, prognosis, treatment, and molecular mechanism studies.

[0003] The main strategy for complete mass spectrometry-based glycopeptide analysis is the bottom-up strategy, as it can simultaneously obtain multiple information such as protein, glycosylation modification sites, and glycan composition. This strategy includes four parts: sample preparation, glycopeptide enrichment, mass spectrometry analysis, and data processing. However, many challenges have been identified during the implementation of this strategy, including microscopic and macroscopic heterogeneity of glycosylation modifications, very low abundance of glycosylation modifications, the need for efficient enrichment strategies during sample preparation to avoid interference from non-glycoproteins or non-glycopeptides, low glycan ionization efficiency during mass spectrometry detection, and complex glycopeptide spectra, necessitating innovation and development at every stage.

[0004] N-glycosylation is a crucial post-translational modification of proteins in eukaryotes, primarily occurring within the endoplasmic reticulum and Golgi apparatus. Approximately 200 glycosyltransferases are involved, contributing to the glycosylation of over 50% of proteins. N-glycosylation involves the attachment of sugar chains to asparagine residues within the conserved NXT / S sequence within the polypeptide chain, catalyzed by glycosyltransferases located on the surface of the endoplasmic reticulum. These sugar chains then enter the endoplasmic reticulum and Golgi apparatus for further processing, extending the common pentasaccharide core to form high-mannose, complex, and hybrid forms. N-glycosylation, in various ways (glycosylated proteins, glycosylated lipids, and glycosylated RNA), is widely involved in fundamental biological processes, including gene transcription, protein translation, protein folding, signal transduction, functional localization, intracellular trafficking, and cell adhesion.

[0005] Currently, there are relevant methods for the analysis of N-glycopeptides obtained after N-glycosylation modification. For example, for mass spectrometry analysis of N-glycopeptides, traditional fragmentation techniques such as sceHCD and EThcD can be used for data acquisition, or new modes such as EThcD-sceHCD (Zhang Y, Zheng S, Mao Y, et al. Systems analysis of plasma IgG intact N-glycopeptides from patients with chronic kidney diseases via EThcD-sceHCD-MS / MS.2021.) can be used for data acquisition.

[0006] For the qualitative identification of N-glycopeptides, analysis can be performed using software such as Byonic and pGlyco. For quantitative analysis, several proteomics data quantification tools already exist, such as PANDA and MaxQuant. However, because the mass spectrometry data of N-glycopeptides differs from that of other proteins in terms of data characteristics, with the presence of triple information on sugar chains, sugar modification sites, and peptide segments, the calculations are complex and computationally intensive. These existing proteomics data quantification tools cannot be directly applied to the quantitative analysis of N-glycopeptide mass spectrometry data. Therefore, the quantitative analysis of N-glycopeptides remains extremely challenging. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the present invention provides a method and system for quantitative analysis of intact N-glycopeptides, aiming to achieve accurate quantitative analysis of intact N-glycopeptides.

[0008] A method for quantitative analysis of intact N-glycopeptides, comprising the following steps:

[0009] Step 1: Pre-treat the glycoprotein sample and perform mass spectrometry detection to obtain N-glycopeptide mass spectrometry data;

[0010] Step 2: Use search library software to perform qualitative analysis of complete N-glycopeptides;

[0011] Step 3: PANDA software was used for complete N-glycopeptide quantitative analysis. The PANDA software parameters were set as follows:

[0012] (1) The number of isotope peaks detected is set according to the intensity of the parent ion signal;

[0013] (2) When constructing the XIC of glycosylated peptides, the XIC cutoff time is 1-3 minutes.

[0014] Preferably, in step 1, the glycoprotein sample pretreatment steps include: protein extraction, denaturation, reduction, alkylation, protease cleavage and complete N-glycopeptide enrichment.

[0015] Preferably, in step 1, the N-glycopeptide mass spectrometry data is acquired using a mass spectrometry detection method based on the EThcD-sceHCD fragmentation mode.

[0016] Preferably, in step 2, the library search software is selected from Byonic or pGlyco.

[0017] Preferably, the PANDA software parameters are set as follows:

[0018] (1) When the parent ion intensity is less than or equal to 1×e8, 3-4 isotope peaks are detected; when the parent ion intensity is greater than 1×e8, 6 isotope peaks are detected;

[0019] (2) When constructing the XIC of glycosylated peptides, the cutoff time of XIC was 2 minutes.

[0020] The present invention also provides a complete N-glycopeptide quantitative analysis system (named N-glycoQuant), comprising:

[0021] Mass spectrometry acquisition module, used to obtain N-glycopeptide mass spectrometry data through mass spectrometry detection;

[0022] Qualitative analysis module, used for qualitative analysis of complete N-glycopeptides using library search software;

[0023] The quantitative analysis module is used to perform complete N-glycopeptide quantitative analysis using PANDA software. The PANDA software parameters are set as follows:

[0024] (1) The number of isotope peaks detected is set according to the intensity of the parent ion signal; (2) When constructing the XIC of the glycosylated peptide segment, the cutoff time of the XIC is 1-3 minutes.

[0025] Preferably, in the mass spectrometry acquisition module, a mass spectrometry detection method based on the EThcD-sceHCD fragmentation mode is used to acquire the N-glycopeptide mass spectrometry data.

[0026] Preferably, in the qualitative analysis module, the library search software is selected from Byonic or pGlyco.

[0027] Preferably, in the quantitative analysis module, the PANDA software parameters are set as follows:

[0028] (1) When the parent ion intensity is less than or equal to 1×e8, 3-4 isotope peaks are detected; when the parent ion intensity is greater than 1×e8, 6 isotope peaks are detected;

[0029] (2) When constructing the XIC of glycosylated peptides, the cutoff time of XIC was 2 minutes.

[0030] The present invention also provides a computer-readable storage medium storing a computer program for implementing the above-mentioned complete N-glycopeptide quantitative analysis method or a computer program for implementing the above-mentioned complete N-glycopeptide quantitative analysis system.

[0031] In the present invention, the "intact N-glycopeptide qualitative analysis" refers to the qualitative analysis of sugar chains, sugar modification sites and peptide segments, and the "intact N-glycopeptide quantitative analysis" refers to the quantitative analysis of glycosylated peptide segments (including sugar chains and peptide segments).

[0032] This invention provides a complete N-glycopeptide mass spectrometry data quantification solution for the quantitative analysis of intact N-glycopeptides. By optimizing the quantitative analysis software parameters based on the characteristics of N-glycopeptide mass spectrometry data, this invention enables large-scale quantitative analysis of intact N-glycopeptides in clinical samples. The methods and systems of this invention enable the identification of differentially expressed intact N-glycopeptides, providing a new approach and strategy for the discovery of novel disease-related intact N-glycopeptide biomarkers.

[0033] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0034] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of a flow chart of an embodiment of the present invention. DETAILED DESCRIPTION

[0036] It should be noted that the algorithms for data collection, transmission, storage and processing steps not specifically described in the embodiments, as well as the hardware structures, circuit connections, etc. not specifically described can all be implemented through the disclosed content of the prior art.

[0037] The reagents and materials used in the following examples, unless otherwise specified, are commercially available.

[0038] Example 1: Intact N-glycopeptide Quantitative Analysis Method and System

[0039] The system of this embodiment includes:

[0040] Mass spectrometry acquisition module, used to obtain N-glycopeptide mass spectrometry data through mass spectrometry detection;

[0041] Qualitative analysis module, used for qualitative analysis of complete N-glycopeptides using library search software;

[0042] The quantitative analysis module is used to perform complete N-glycopeptide quantitative analysis using PANDA software. The PANDA software parameters are set as follows:

[0043] (1) When the parent ion intensity is less than or equal to 1×e8, 3-4 isotope peaks are detected; when the parent ion intensity is greater than 1×e8, 6 isotope peaks are detected;

[0044] (2) When constructing the XIC of glycosylated peptides, the cutoff time of XIC was 2 minutes.

[0045] The process of quantitative analysis of intact N-glycopeptides using the above system is as follows: Figure 1 As shown, the specific steps include:

[0046] 1. Glycoprotein Sample Pretreatment

[0047] 1. Thaw plasma and prepare buffer: 20 μl of plasma contains approximately 200-300 μg of IgG. The binding buffer consists of 25 mM Tris, 150 mM NaCl, pH 7.2, and the elution buffer consists of 0.1 M formic acid.

[0048] 2. Take 20uL of immobilized Protein A / G material, centrifuge at room temperature, discard the supernatant, and wash twice with binding buffer.

[0049] 3. Dilute the plasma sample to 200ul with binding buffer.

[0050] 4. Rotate and mix at room temperature for 2 h.

[0051] 5. Centrifuge at 3000 g to remove the supernatant and wash three times with 500 μl of binding buffer, rotating for 5 minutes each time.

[0052] 6. Add 50 μl of 0.1 M formic acid for elution, mix at room temperature for 5 minutes, and elute 10 times in total. Centrifuge and collect the supernatant.

[0053] 7. Use Bradford method to quantify glycoprotein IgG.

[0054] 8. Take 20 μg of IgG and add it to 150 μl of ammonium bicarbonate solution (pH 8.5).

[0055] 9. Denature by heating at 95°C for 10 minutes.

[0056] 10. Add 4 μl of 1 M DTT and reduce the reaction at 56 degrees for 45 minutes.

[0057] 11. Add 10 μl of 1 M IAM and allow the alkylation reaction to proceed in the dark at room temperature for 30 min.

[0058] 12. Transfer to a 10KDa ultrafiltration tube, wash three times with ammonium bicarbonate solution, centrifuge at 13000g for 10min and replace with a new tube.

[0059] 13. Add 500 ng of trypsin and shake overnight.

[0060] 14. Centrifuge at 13,000 g for 10 min. Wash three times with 70 μl of ultrapure water each time. Collect the peptides and store them at -80°C.

[0061] 15. Dissolve the peptide fragment in 100 μL of equilibrium solution (80% ACN, 0.2% TFA in H2O).

[0062] 16. Weigh 20 mg of ZIC-HILIC and activate it with 0.1% TFA three times, each time for 10 minutes.

[0063] 17. Equilibrate ZIC-HILIC with 200 μL of equilibration solution for 5 minutes each time, three times.

[0064] 18. Remove the equilibration solution and add ZIC-HILIC to the sample. Rotate at room temperature for 2 hours.

[0065] 19. Prepare a C8 membrane and place one layer into a 200 μL pipette tip. Place the sample into the tip and centrifuge at 3000 g for 1 min. Wash three times with 70 μL of equilibration solution.

[0066] 20. Replace the EP tube with a new one, add 70uL 0.1% TFA and wash three times, collect the intact N-glycopeptide and heat dry it.

[0067] 2. Mass spectrometry analysis

[0068] 1. Analyze using Orbitrap Fusion Lumos Tribrid mass spectrometry.

[0069] 2. The sample was dissolved in 0.1% FA solution and then separated using a reverse phase chromatography direct injection column Magic C18 column (Pur C18-AQ filler diameter 1.9 μm, inner diameter 75 μm, column length 20 cm, Dr Maisch).

[0070] 3. Chromatographic conditions: mobile phase A (0.1% FA), mobile phase B (80% ACN, 0.1% FA), mobile phase B linear gradient of 6-32%, separation gradient of 78 min, flow rate of 0.3 μL / min.

[0071] 4. The EThcD-sceHCD-MS / MS mass spectrometry conditions are divided into two parts of the experiment.

[0072] 5. Experiment 1 (EThcD): The primary spectrum ion scan mass range was 400-1600 m / z, the resolution was 60,000, the RFlens was 30%, the AGC target was custom, the maximum injection time was 50 ms, the exclusion time was 15 s, and the cycle time was 2 s; the secondary mass spectrometry separation window was 2 m / z, the Orbitrap resolution was 30,000, the AGC target was custom, the maximum injection time was 150 ms, and the EThcD fragmentation energy was 35%.

[0073] 6. Experiment 2 (sceHCD): The primary spectrum ion scan mass range was 400-1600 m / z, the resolution was 60,000, the RF lens was 30%, the AGC target was standard, the maximum injection time was auto, the exclusion time was 15 s, and the cycle time was 2 s. The secondary mass spectrometry separation window was 1.6 m / z, the Orbitrap resolution was 30,000, the AGC target was 200%, the maximum injection time was auto, and the HCD step fragmentation energy was 20-30-40%.

[0074] 3. Identification of intact N-glycopeptides

[0075] 1. The RAW files (EThcD-sceHCD) obtained by mass spectrometry analysis were searched using Byonic software (v3.6.0, ProteinMerics, Inc.) using the IgG protein amino acid sequence database.

[0076] 2. Search parameter settings: The mass error of the precursor ion peptide segment in the primary mass spectrometry scan was 10 ppm, the mass error of the fragment ion peptide segment in the secondary mass spectrometry scan was 20 ppm, the enzyme digestion method was trypsin and / or Glu-C, and the maximum number of missed cleavage sites was 2. The fixed modification was set to carboxyamidomethylation (C); the variable modifications were set to acetyl (protein N-term), oxidation (M), and deamidation (N). In addition, for the analysis of N-glycosylation modifications of human plasma IgG, 182 human N-glycans were set as variable modifications. The fragmentation mode was set to EThcD-HCD; and the false positive rate for protein and peptide matching was set to 1%.

[0077] 3. Leave other parameters as default and click Run.

[0078] 4. Quantification of Intact N-Glycopeptides

[0079] 1. The identification results obtained by Byonic software analysis were quantified using PANDA software. The PANDA software parameters were set as follows: (1) When performing peak detection, the number of isotope peaks detected was automatically set according to the parent ion signal intensity (rule: when the parent ion intensity was less than or equal to 1×e8, 3-4 isotope peaks were detected; when the parent ion intensity was greater than 1×e8, 6 isotope peaks were detected). This helped to more sensitively detect the parent ions corresponding to the glycosylated peptides (usually the parent ion intensity was not high); (2) When constructing the XIC of the glycosylated peptides for quantification, the XIC cutoff time was optimized to 2 minutes based on the data characteristics, which helped to improve the accuracy of peak quantification.

[0080] 2. First select the quantity according to the RAW files, and then import the RAW files into the software.

[0081] 3. After each RAW file corresponding to the Byonic search result output.spectra file is input into the software one by one.

[0082] 4. Select the output location for the results.

[0083] 5. Click the software to run.

[0084] The above method can be used to complete the quantitative analysis of complete N-glycopeptides.

[0085] As demonstrated in the examples above, the present invention provides a comprehensive N-glycopeptide mass spectrometry data quantification solution for the quantitative analysis of intact N-glycopeptides. This method can be used for large-scale quantitative analysis of intact N-glycopeptides in clinical samples and for the discovery of novel biomarkers. It holds great promise for drug development and the research of novel disease-related intact N-glycopeptide biomarkers.

Claims

1. A method for quantitative analysis of intact N-glycopeptides, characterized in that: The steps include: Step 1: Pre-treat the glycoprotein sample and perform mass spectrometry detection to obtain N-glycopeptide mass spectrometry data; Step 2: Use search library software to perform qualitative analysis of complete N-glycopeptides; Step 3: PANDA software was used for complete N-glycopeptide quantitative analysis. The PANDA software parameters were set as follows: (1) The number of isotope peaks detected is set according to the parent ion signal intensity; when the parent ion intensity is less than or equal to 1×e8, 3-4 isotope peaks are detected, and when the parent ion intensity is greater than 1×e8, 6 isotope peaks are detected; (2) When constructing the XIC of glycosylated peptides, the XIC cutoff time was 2 minutes; In step 1, the glycoprotein sample pretreatment steps include: protein extraction, denaturation, reduction, alkylation, protease cleavage and complete N-glycopeptide enrichment; In step 1, the mass spectrometry data of the N-glycopeptide is acquired by using a mass spectrometry detection method based on the EThcD-sceHCD fragmentation mode; In step 2, the library search software is selected from Byonic or pGlyco.

2. A complete N-glycopeptide quantitative analysis system, characterized in that: include: Mass spectrometry acquisition module, used to obtain N-glycopeptide mass spectrometry data through mass spectrometry detection; Qualitative analysis module, used for qualitative analysis of complete N-glycopeptides using library search software; The quantitative analysis module is used to perform complete N-glycopeptide quantitative analysis using PANDA software. The PANDA software parameters are set as follows: (1) The number of isotope peaks detected is set according to the parent ion signal intensity; when the parent ion intensity is less than or equal to 1×e8, 3-4 isotope peaks are detected, and when the parent ion intensity is greater than 1×e8, 6 isotope peaks are detected; (2) When constructing the XIC of glycosylated peptides, the XIC cutoff time was 2 minutes; In the mass spectrometry acquisition module, the mass spectrometry detection method based on the EThcD-sceHCD fragmentation mode is used to acquire the N-glycopeptide mass spectrometry data; In the qualitative analysis module, the library search software is selected from Byonic or pGlyco.

3. A computer-readable storage medium, characterized in that: A computer program for implementing the method for quantitative analysis of intact N-glycopeptides according to claim 1 or a computer program for implementing the system for quantitative analysis of intact N-glycopeptides according to claim 2 is stored thereon.

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