High-throughput targeted mass spectrometry method for site-specific N-glycopeptide

Abstract

The invention belongs to the technical field of biological analysis, and relates to an N-glycopeptide high-throughput targeted mass spectrometry analysis method, in particular to a site-specific complete N-glycopeptide high-throughput targeted mass spectrometry analysis method. The method comprises the following steps: carrying out two-step derivation on a site-specific complete N-glycopeptide by using an equal-weight isotope tag TMT reagent and light and heavy isotope isopropylamine, and then carrying out data acquisition by using a targeted mass spectrometry method. According to the method disclosed by the invention, the change of the relative content of the site-specific complete N-glycopeptide in 20 samples can be analyzed at the same time in each time of mass spectrum sampling. According to the analysis method disclosed by the invention, rapid treatment and accurate analysis of a complete N-glycopeptide sample are realized by introducing a chemical derivation step of isotope labeling.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological analysis, and relates to a method for high-throughput targeted mass spectrometry analysis of N-glycopeptides, and specifically to a method for high-throughput targeted mass spectrometry analysis of site-specific intact N-glycopeptides, wherein the method achieves rapid processing and accurate analysis of intact N-glycopeptide samples by introducing an isotope-labeled chemical derivatization step. Background Art

[0002] The prior art discloses that mass spectrometry is an important technology in the field of bioanalysis. However, practice shows that traditional mass spectrometry methods have certain limitations, especially in high-throughput analysis. As a powerful tool, targeted mass spectrometry methods have high accuracy and high sensitivity, but they can usually only analyze one sample at a time, which greatly limits the analysis efficiency and sample detection throughput.

[0003] Intact N-glycopeptides are an important class of biomarkers that are of great significance for the diagnosis and treatment of diseases. However, existing methods for analyzing intact N-glycopeptides can usually only analyze one sample per mass spectrometry analysis, which is limited by the sample analysis throughput and has low analysis efficiency.

[0004] Site-specific intact glycopeptides are an important class of biomarkers, but due to the lack of corresponding antibodies, targeted mass spectrometry analysis is usually required. Although the existing targeted mass spectrometry analysis method has high selectivity and sensitivity for analyzing site-specific glycopeptides, its throughput still needs to be further improved.

[0005] Therefore, there is an urgent need in this technical field to develop a high-throughput targeted mass spectrometry analysis method that can quickly and accurately verify a large sample size of intact N-glycopeptide samples, thereby accelerating the discovery, verification and clinical application of biomarkers.

[0006] Based on the current status and existing problems of the prior art, the inventors of this application propose a new high-throughput targeted mass spectrometry analysis method for site-specific intact N-glycopeptides. This method introduces a chemical derivatization step. By using isobaric isotope labels and light and heavy isotope isopropylamine to chemically derivatize site-specific intact N-glycopeptides, rapid processing and accurate analysis of intact N-glycopeptide samples are achieved, and the sensitivity and accuracy of the analysis can be effectively improved. Summary of the invention

[0007] The purpose of the present invention is to propose a new site-specific intact N-glycopeptide high-throughput targeted mass spectrometry analysis method based on the current status and existing problems of the prior art. The method introduces a chemical derivatization step. By using isobaric isotope labels and light and heavy isotope isopropylamine to chemically derivatize site-specific intact N-glycopeptides, rapid processing and accurate analysis of intact N-glycopeptide samples are achieved, and the sensitivity and accuracy of the analysis can be effectively improved.

[0008] The present invention is aimed at the industry's relevant site-specific intact glycopeptides as an important class of biomarkers, but due to the lack of corresponding antibodies, targeted mass spectrometry analysis is usually required. Although the current targeted mass spectrometry analysis method has high selectivity and sensitivity for analyzing site-specific glycopeptides, its throughput still needs to be further improved. Therefore, the present invention provides a method for high-throughput targeted mass spectrometry analysis of site-specific intact N-glycopeptides, which aims to overcome the limitation that traditional targeted mass spectrometry analysis methods can only analyze one sample at a time, and provides a fast and accurate analysis method. The method utilizes chemical derivatization so that the site-specific intact N-glycopeptides in different samples can produce parent ion fragments with different mass numbers or daughter ion fragments for quantification after chemical derivatization, thereby achieving simultaneous detection in the mass spectrometry and improving the analysis throughput. The method of the present invention has broad application prospects and can be used for clinical diagnosis and treatment, as well as biomedical research. It provides a reliable solution for the development of research and application of glycopeptides as biomarkers, and promotes progress in the fields of medicine and biomedicine. In order to achieve the above-mentioned invention purpose, the present invention adopts the following technical scheme:

[0009] a. Perform two-step derivatization of site-specific intact N-glycopeptides with isobaric TMT labeling and light and heavy isotopic isopropylamine;

[0010] b. Using targeted mass spectrometry analysis method for data acquisition;

[0011] c. Each mass spectrometry injection simultaneously analyzes the changes in the relative content of site-specific intact N-glycopeptides in 20 samples.

[0012] Specifically, the present invention provides a novel glycopeptide derivatization method, which includes purifying immunoglobulin G (IgG) from a sample, and digesting IgG with trypsin to produce site-specific glycosylated peptides. Subsequently, the N-terminus and C-terminus of the peptides are labeled with an isotope label TMT and a light and heavy isotope isopropylamine chemical derivatization reagent, respectively.

[0013] The introduction of the chemical derivative label can not only improve the sensitivity of the analysis, but also simplify the complexity of the chromatogram, and realize high-throughput parallel reaction monitoring targeted mass spectrometry (PRM-MS) analysis for site-specific complete N-glycopeptides. In a single experiment, the present invention can analyze up to 20 samples simultaneously by combining the quantitative information of the primary mass spectrometry and the secondary mass spectrometry. One channel in each ten-mark TMT labeling reagent is used as an internal reference group, so the relative abundance of complete N-glycopeptides in each sample can be determined simultaneously based on the intensity of the TMT reporter ion. Compared with the current conventional targeted mass spectrometry method that can only analyze a single sample at a time, the method of the present invention expands the detection channel by 20 times, and can analyze up to 480 samples per day (24 hours × 20), and each sample only takes 3 minutes on average. In the application of the present invention, the method is used to analyze the relative content of complete N-glycopeptides in serum IgG of patients with liver disease, and combined with differential analysis, the changes in IgG complete N-glycopeptides related to the development of liver disease are confirmed. It is expected that the classification model of corresponding severe liver diseases will be established through machine learning based on the results. The method invented by benzene not only provides an innovative and efficient method for the targeted verification of site-specific intact N-glycopeptides in the serum of liver cancer patients, but also has the potential to serve as a powerful diagnostic tool for biomarkers of other diseases in the future.

[0014] More specifically,

[0015] The present invention provides a method for high-throughput targeted mass spectrometry analysis of site-specific N-glycopeptides, comprising the following steps: (1) collecting serum samples; (2) purifying immunoglobulin G (IgG) in the serum and performing enzymatic hydrolysis; (3) performing derivatization treatment using an isobaric isotope label 10-plex TMT; (4) enriching IgG complete N-glycopeptides; (5) performing light / heavy isopropylamine derivatization treatment; (6) performing high-throughput detection using LC-PRM-MS / MS; and (7) performing data processing and differential analysis.

[0016] Preferably, the step (1): collecting a serum sample and storing it in a refrigerator at -80°C to ensure the preservation quality of the sample;

[0017] Preferably, the step (2) comprises:

[0018] (a) Serum samples were thawed at 4°C;

[0019] (b) IgG protein was purified from serum using protein G-agarose;

[0020] (c) The concentration of purified IgG protein was determined using a commercially available BCA quantification kit (Thermo Fisher Scientific).

[0021] (d) Take 100 μg of protein from each sample described in (3) and dilute it to 1 μg / μL with 100 mM tetraethylammonium bromide (TEAB).

[0022] (e) Reduction alkylation of IgG protein: Add dithiothreitol (DTT) at a final concentration of 15 mM to 100 μg of protein, and perform reduction reaction at 56°C for 30 min. Then add iodoacetamide (IAA) at a final concentration of 30 mM to the above mixture for alkylation reaction, and keep at room temperature in the dark for 45 min.

[0023] (f) Methanol-chloroform precipitation: The IgG protein solution after reduction and alkylation is subjected to methanol-chloroform precipitation to remove excess DTT and IAA. Add the reagents according to the solution volume ratio of 1:4:1:3 (protein solution: methanol: chloroform: water), shake and mix, and then centrifuge (15000rcf, 20℃, 20min). Discard the upper solution and place it in a fume hood to air dry for 2-3min before enzymatic hydrolysis.

[0024] (g) IgG proteolysis: 100 μL 100 mM TEAB was added, and trypsin was added at a mass ratio of 1:50 (enzyme:protein), and the enzymatic digestion was carried out at 37°C overnight. The next day, trifluoroacetic acid (TFA, final concentration of 0.5%) was added to the peptide solution after enzymatic digestion to terminate the enzymatic digestion. Excess salt was removed by C18 desalting column (50 mg, Waters) and then dried on a rotary vacuum concentrator (Christ, Germany).

[0025] Preferably, step (3) comprises: performing a TMT labeling procedure (e.g., the method of ThermoScientific) according to the manufacturer's instructions. That is, the IgG peptides in all samples are subjected to TMT labeling treatment according to the manufacturer's instructions (Thermo Fisher Scientific). Specifically, 8 μL of TMT reagent with different labels are added to 100 μg of peptides after enzymatic hydrolysis, and the reaction is shaken at room temperature for 1 hour. After the reaction is completed, 1.6 μL of 5% hydroxylamine solution is added to each tube to consume the unreacted TMT labeling reagent. Finally, the 10-plex TMT-labeled enzymatic peptides are mixed according to the required ratio, stored in a -80°C refrigerator, and wait for HILIC enrichment. TMT-131 is used as an internal standard reference group for labeling to correct batch effects and eliminate errors caused by different operations or instruments. Two samples are selected from each of the healthy control group, hepatitis group, cirrhosis group, and liver cancer group, and the other 8 TMT channels are labeled. The last TMT channel randomly selects a sample from all samples for labeling;

[0026] Preferably, step (4) comprises: the method of enriching intact N-glycopeptides using HILIC is as follows: first, the HILIC filler is loaded into a 200 μL pipette tip according to a ratio of protein mass to HILIC filler of 1:60. Next, the HILIC column is activated 3-4 times with 200 μL activation solution (0.1% TFA) to ensure the activity of the column. Then, the HILIC column is equilibrated 3-4 times with 200 μL equilibration solution (80% ACN, 1% TFA) to prepare for injection. During the sample processing, the IgG protease peptides dissolved in the equilibration solution are loaded 3-4 times, and then the HILIC column enriched with glycopeptides is washed 3-4 times with the equilibration solution to remove non-glycopeptide substances that are non-specifically bound. Finally, the intact N-glycopeptides enriched on the HILIC column are eluted 3 times with 100 μL 0.1% TFA eluent, and the eluents are collected and combined, and then dried by vacuum centrifugation for standby use. Through the above steps, HILIC fillers can be used for effective enrichment of intact N-glycopeptides. First, the column is activated and equilibrated to ensure the performance of the column and the state of being ready for injection. Subsequently, through multiple sample loading and washing steps, non-glycopeptide substances are removed and the enriched intact N-glycopeptides are retained. Finally, the intact N-glycopeptides are eluted from the column through the elution step and vacuum centrifuged for subsequent derivatization or storage. The optimization of this enrichment step can improve the detection sensitivity and accuracy of intact N-glycopeptides, and can effectively remove interfering substances, thereby obtaining more reliable results;

[0027] Preferably, step (5) comprises: labeling the glycopeptide with isopropylamine. The 10 mixed samples labeled with TMT are respectively mixed with 20 μL of isopropylamine labeling solution (composed of NMM:DMSO (3:7) mixed solution, 1M EDC-HCl, 1M HoBt and 4M light-labeled isopropylamine (iPA-H7)). The samples are incubated at 25°C (room temperature) for 4 hours to complete the labeling process. At the same time, another 10 samples are treated in the same way, but are subsequently labeled with heavy-labeled isopropylamine (iPA-D7). After the labeling is completed, the two groups of samples are remixed, and excess reagents and impurities are removed by HILIC, and then freeze-dried. Through the processing of this step, a double-labeled sample can be obtained for subsequent mass spectrometry analysis and data processing;

[0028] Preferably, step (6) comprises: the experiment uses Thermo Fisher Scientific's EASY-NanoLC1200 system in conjunction with an Orbitrap Exploris480 mass spectrometer. First, the glycopeptide is loaded onto an analytical column (AcclaimPepMap C18, 75 μm×25 cm) and separated using a linear gradient elution. Solvent A is water containing 0.1% FA, and solvent B is a solution containing 80% ACN and 0.1% FA. A total of 60 minutes of gradient separation was performed for the intact N-glycopeptide in the IgG sample, and the specific separation gradient is as follows: from 0 to 1 minute, the gradient is 2-8%, from 1 to 31 minutes, the gradient is 8-18%, from 31 to 35 minutes, the gradient is 18-30%, from 35 to 55 minutes, the gradient is 30-55%, and finally maintained at 95% level for 5 minutes. The column flow rate is maintained at 300 nL / min. The parameters for glycopeptide analysis were set as follows: MS1 scan: (1) Scan range (m / z) = 400-2000; resolution = 60,000; normalized AGC target value (%) = 300; maximum injection time = 50 ms; included charge states = 2-6; dynamic exclusion times n = 1; dynamic exclusion time = 15 s; HCD-MS / MS was performed once for each selected precursor ion. (2) HCD-MS / MS parameters: isolation window = 2.2; detector type = Orbitrap; resolution = 15,000; normalized AGC target value (%) = 300; maximum injection time = 50 ms; collision energy (%) = 20, 30, 40. Through the above settings, efficient separation and mass spectrometry analysis of glycopeptides can be achieved to obtain accurate data and results;

[0029] Preferably, step (7) comprises: for the relative contents of IgG intact N-glycopeptides between patients with three types of liver diseases (hepatitis, cirrhosis and liver cancer) and healthy controls obtained by high-throughput targeted mass spectrometry detection method, using R (version 4.2.2) to perform significant difference analysis on the relative contents of IgG intact N-glycopeptides using one-way analysis of variance (ANOVA). This step can use statistical methods to compare the contents of IgG intact N-glycopeptides between different groups to reveal significant differences between liver disease and healthy state. This helps to assess the degree of deterioration of liver disease and provides important guidance and reference for the early diagnosis and treatment of related diseases.

[0030] Technical effects of the present invention:

[0031] High throughput: The present invention uses isobaric isotope label 10-plex TMT and double-label light / heavy isopropylamine labeling methods to achieve simultaneous analysis of up to 20 samples in a single experiment, and up to 480 samples can be analyzed per day (24 hours × 20). This high throughput greatly improves sample processing efficiency and is suitable for glycopeptide analysis of large-scale sample sets.

[0032] High efficiency: It takes only 3 minutes to complete the mass spectrometry test of each sample on average, which greatly shortens the sample detection time. This high efficiency makes the analysis process faster and improves the sample detection throughput of the experiment.

[0033] High selectivity and sensitivity: The method of the present invention combines the quantitative information of TMT reporter ions in primary mass spectrometry and tandem mass spectrometry, and can simultaneously determine the relative abundance of intact IgG N-glycopeptides in each sample with high selectivity and sensitivity. Through precise quantitative analysis, it can accurately detect and quantify site-specific glycopeptide changes, providing important information for studying the development of the disease.

[0034] Strong application potential: The method of the present invention is efficient and accurate in identifying glycopeptide changes associated with the disease development process, and provides a reliable data basis for establishing corresponding classification models. This application potential makes the method of the present invention a powerful tool that can provide a new biomarker verification method for disease diagnosis and treatment. By studying the changes in glycopeptides under different disease states, more in-depth and comprehensive information can be provided for biomedical research, clinical diagnosis and treatment, and other fields, promoting scientific progress and the development of clinical applications.

[0035] Therefore, the technical effects of the present invention include high throughput, high efficiency, high selectivity and sensitivity, and great application potential.

[0036] Compared with the traditional mass spectrometry analysis method, the method of the present invention has significant advantages. First, the method can simultaneously analyze the changes in the relative content of site-specific intact N-glycopeptides in 20 samples per mass spectrometry injection, achieving high-throughput sample analysis. Secondly, the introduced chemical derivatization step greatly simplifies the complexity of the chromatogram and improves the analysis efficiency and accuracy. In addition, this method is not only suitable for the analysis of intact N-glycopeptides, but can also be applied to a wide range of other sample types, with high flexibility and applicability.

[0037] In summary, the present invention provides an innovative high-throughput targeted mass spectrometry analysis method, which achieves effective processing and accurate analysis of intact N-glycopeptide samples through chemical derivatization. This method has broad prospects and application value in biomarker validation and clinical application, and has brought important technological breakthroughs in the field of chemical derivatization bioanalysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is the MALDI-MS spectrum of the intact N-glycopeptide of IgG, showing the derivatization efficiency of TMT and light / heavy isopropylamine labeling evaluated using the intact N-glycopeptide of IgG. The MALDI-MS spectrum of the intact N-glycopeptide of IgG: a. no derivatization, b. only TMT derivatization labeling, c. TMT and iPA-H7 labeling, d. TMT and iPA-D7 labeling; the red and blue shadows represent the m / z peaks of IgG1-H4N4F1 and IgG1-H5N4F1S1, respectively.

[0039] Figure 2 The sample pretreatment method for verifying intact N-glycopeptides by high-throughput targeted mass spectrometry was used to describe and statistically analyze the intact N-glycopeptides of IgG in three liver disease patient groups and a healthy control group. The bubble chart showed the relative abundance of 32 target intact N-glycopeptides in the four sample groups, where H represents healthy people, M represents hepatitis patients, Y represents cirrhosis patients, and C represents liver cancer patients. DETAILED DESCRIPTION

[0040] Example 1 Collection of serum from 461 patients (including patients with liver cancer / cirrhosis / hepatitis and healthy subjects) and sample pre-treatment of IgG intact N-glycopeptide:

[0041] Serum samples were collected from 127 patients clinically diagnosed with hepatitis, 106 patients with cirrhosis, 108 patients with liver cancer, and 120 healthy subjects.

[0042] Main instruments and reagents: EP tube, BCA quantitative kit (Thermo Fisher Scientific), 10-plex TMT labeling reagent (Thermo Fisher Scientific), light / heavy isopropylamine reagents, 1-hydroxybenzotriazole (HoBt), PBS buffer solution, N-methylmorpholine (NMM), EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide), DMSO (dimethyl sulfoxide), DTT (dithiothreitol), IAA (indoleacetic acid), trypsin, etc.

[0043] Experimental steps:

[0044] 1. Collection of serum samples:

[0045] (1) Collect blood samples from clinical patients and separate serum. Store the serum samples in a -80℃ refrigerator.

[0046] 2. Purification and enzymatic hydrolysis of serum immunoglobulin G (IgG):

[0047] (1) Thaw serum samples at 4°C.

[0048] (2) IgG protein was purified from serum using protein G-agarose.

[0049] (3) The concentration of the purified IgG protein was measured using a commercially available BCA quantification kit (Thermo Fisher Scientific).

[0050] (4) Take 100 μg of protein from each sample and dilute it to a concentration of 1 μg / μL using 100 mM tetraethylammonium bromide (TEAB).

[0051] (5) Reductive alkylation of IgG protein: Add dithiothreitol (DTT) at a final concentration of 15 mM to 100 μg of protein, and perform reduction reaction at 56°C for 30 minutes. Then, add iodoacetamide (IAA) at a final concentration of 30 mM to the mixture, and perform alkylation reaction at room temperature in the dark for 45 minutes.

[0052] (6) Methanol-chloroform precipitation: The reduced alkylated IgG protein solution was mixed with methanol-chloroform in a volume ratio of 1:4:1:3, shaken and mixed, and then centrifuged (15000rcf, 20°C, 20 minutes). After removing the upper layer of solution, it was naturally air-dried in a fume hood for 2-3 minutes, and then enzymatic hydrolysis was performed.

[0053] (7) Enzymatic hydrolysis of IgG protein: 100 μL of 100 mM TEAB was added to the peptide solution after enzymatic hydrolysis, and trypsin was added at a mass ratio of 1:50 (enzyme: protein), and the enzymatic hydrolysis was carried out at 37°C overnight. The next day, trifluoroacetic acid (TFA, final concentration of 0.5%) was added to the peptide solution after enzymatic hydrolysis to terminate the enzymatic hydrolysis. Excess salt was removed using a C18 desalting column (50 mg, Waters), and then the sample was dried using a rotary vacuum concentrator (Christ, Germany).

[0054] 3. Pre-treatment of IgG intact N-glycopeptide samples:

[0055] (1) TMT labeling of intact IgG N-glycopeptides: TMT labeling was performed on IgG peptides in all samples according to the manufacturer's instructions (Thermo Fisher Scientific). The specific steps are as follows: 8 μL of different labeled TMT reagents were added to each 100 μg peptide after enzymatic digestion, and the reaction was shaken at room temperature for 1 hour. After the reaction, 1.6 μL of 5% hydroxylamine solution was added to each tube to consume the incompletely reacted TMT labeling reagent. Finally, the 10-plex TMT-labeled enzymatic peptides were mixed according to the required ratio and stored in a -80°C refrigerator for HILIC enrichment.

[0056] (2) ZIC-HILIC enrichment of intact glycopeptides: First, prepare a ZIC-HILIC column and plug a 200 μL pipette tip with cotton. Weigh 30 mg of ZIC-HILIC chromatographic filler into a 200 μL pipette tip, then centrifuge at 3000 rcf to compact the filler. Use 200 μL of 0.1% TFA solution to activate the ZIC-HILIC column, centrifuge at 2500 rcf for 2 minutes, and repeat twice. Use 200 μL of 80% ACN 1% TFA solution to equilibrate the ZIC-HILIC column, centrifuge at 3500 rcf for 1 minute, and repeat 3 times. Add the 80% ACN 1% TFA solution to the TMT-labeled IgG peptide and add it to the ZIC-HILIC column. Centrifuge at 3500 rcf for 90 seconds to complete the loading step. The ZIC-HILIC column was washed with 200 μL 80% ACN 1% TFA solution and centrifuged at 3500 rcf for 1 minute to remove non-glycosylated peptides and non-specific adsorbed substances. This was repeated three times. The intact glycopeptides were eluted with 100 μL 0.1% TFA, which was repeated three times. The collected eluate was lyophilized.

[0057] (3) Subsequently, the 10 glycopeptide samples that had been TMT-labeled and mixed were labeled with isopropylamine. 20 μL of isopropylamine labeling solution (containing a mixed solution of NMM:DMSO (3:7) with a final concentration of 1M EDC-HCl, 1M HoBt, and 4M iPA-H7) was added to the 10 mixed samples labeled with TMT and incubated at 25°C (room temperature) for 4 hours. In addition, another 10 samples were treated using the same method, but with iPA-D7 for subsequent labeling. After labeling, the two sets of mixed samples were remixed and excess reagents were removed by HILIC.

[0058] 4. LC-MS / MS analysis and relative quantification of intact IgG N-glycopeptides:

[0059] Instrument model: The analysis was performed using an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific).

[0060] Chromatographic column model: Acclaim PepMap C18 chromatographic separation column (2.1 μm, 75 μm, 250 mm, ThermoFisher Scientific) was used for sample separation.

[0061] Mobile phase: Use the following gradient conditions for elution:

[0062] Phase A: 0.1% FA (formic acid) solution

[0063] Phase B: 80% ACN (acetonitrile) containing 0.1% FA solution. The elution gradient conditions are:

[0064]

[0065] Mass spectrometry detection conditions:

[0066] Instrument: Orbitrap Exploris 480 mass spectrometer (ThermoFisher Scientific)

[0067]

[0068]

[0069] Example 2: Using high-throughput targeted mass spectrometry to detect the content of intact IgG N-glycopeptides in serum and performing differential analysis on the results

[0070] 1. Import the raw data into Byonic software for data collection:

[0071] (1) Use Byonic software to import raw data for data analysis and processing.

[0072] (2) For IgG samples, the protein database contains four IgG subtypes (IgG1-4), whose corresponding UniProtIDs are P01857, P01859, P01860 and P01861, respectively.

[0073] (3) The tolerance settings for primary and secondary ions are 10 ppm and 20 ppm, respectively.

[0074] (4) The enzyme digestion type was set to trypsin-specific digestion with no missed cuts.

[0075] (5) The charge number is set to 2, 3, 4, 5, and 6.

[0076] (6) Fixed modifications include Cysteine ​​carbamidomethylation.

[0077] (7) Variable modifications include Met (M+15.995Da, rare1) and N-glycosylation (common1). (8) FDR (False Discovery Rate) was controlled at a level of <1%.

[0078] 2. Use R (version 4.2.2) to compare the significant differences in intact N-glycopeptides between different groups: (1) Use R programming language for data analysis and statistical processing.

[0079] (2) The one-way analysis of variance (ANOVA) method was used to analyze the significant differences in the relative contents of all glycopeptides in the four disease groups.

[0080] (3) Based on the statistical results, complete N-glycopeptides with significant differences (p < 0.05) among the four groups were screened as candidate IgG N-glycopeptide biomarkers.

[0081] In Example 2, ensure that accurate and reliable data analysis software and statistical methods are used for data processing and differential analysis. In addition, according to the experimental design and requirements, the parameters and conditions can be appropriately adjusted to obtain the optimal analysis results and ensure the repeatability and reliability of the results.

[0082] Results: Figure 1 The MALDI-MS images shown show the results of investigating the derivatization efficiency of TMT and iPA-H7 / D7 in high-throughput targeted mass spectrometry detection technology. Figure 2 The high-throughput targeted mass spectrometry detection method was used to detect the expression levels of 32 glycopeptides in 461 sera, indicating that the relative contents of the 32 glycopeptides were significantly increased with the increase in the severity of liver diseases (P<0.05).

Claims

1. A method for high-throughput targeted mass spectrometry analysis of site-specific intact N-glycopeptides, characterized in that: include: a. Perform two-step derivatization of site-specific intact N-glycopeptides with isobaric TMT labeling and light and heavy isotopic isopropylamine; b. Using targeted mass spectrometry analysis method for data acquisition; c. Each mass spectrometry injection simultaneously analyzes the changes in the relative content of site-specific intact N-glycopeptides in 20 samples.

2. The method according to claim 1, characterized in that The method comprises the following steps: (1) Collect serum samples; (2) Purify immunoglobulin G (IgG) in serum and perform enzymatic hydrolysis; (3) Derivatize with isotope label 10-plex TMT; (4) Enrich intact IgG N-glycopeptides; (5) Perform light / heavy isopropylamine derivatization; (6) Perform high-throughput detection using LC-PRM-MS / MS; (7) Perform data processing and differential analysis.

3. The method according to claim 1, characterized in that In the described method, isobaric isotopic labeling of site-specific intact N-glycopeptides and targeted mass spectrometry analysis methods are used to achieve high-throughput targeted detection of 20 samples simultaneously in a single mass spectrometry run.

4. The method according to claim 1, characterized in that The described method can be used for the analysis of intact N-glycopeptides, or other sample types including proteins, peptides and / or amino acid metabolites.

5. Use of the method for high-throughput targeted mass spectrometry analysis of site-specific intact N-glycopeptides according to claim 1 in preparing a tool for evaluating the relative content of glycopeptides for different diseases in serum samples.

6. Use of the method for high-throughput targeted mass spectrometry analysis of site-specific intact N-glycopeptides according to claim 1 in the preparation of a high-throughput targeted mass spectrometry analysis tool for 32 intact IgG N-glycopeptides in a large sample size of liver disease serum.

7. Use of the method for high-throughput targeted mass spectrometry analysis of site-specific intact N-glycopeptides according to claim 1 in the preparation of a biomarker validation tool.

8. The use according to claim 6, characterized in that The biomarkers are post-translationally modified protein biomarkers, wherein no reliance on specific antibodies is required.

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