Hydrophilic interaction chromatography coupled with microflow rate colorimetric tandem mass spectrometry for glycoprotein analysis

Abstract

The present application relates to a kind of glycosylated proteomics analysis methods based on hydrophilic interaction chromatography coupling micro liter flow rate's liquid chromatography tandem mass spectrometry.The specific is to use biological sample after by denaturation enzymatic hydrolysis protein obtains peptide, after desalination is submitted to the liquid chromatograph equipped with click maltose microspheres packing hydrophilic interaction chromatography column for enriching the complete glycopeptide in sample, then the sample after enrichment is submitted to micro liter flow rate's liquid chromatography tandem mass spectrometry and carries out the detection of complete glycopeptide.The present application is analyzed glycosylated proteomics of biological sample by certain specification chromatographic column in specific micro liter flow rate's liquid chromatograph, and the reproducibility of analysis method is high, and suitable for long-term stable operation, and this new method of continuous and automatic enrichment of a large number of samples N-glycopeptide has potential application in large-scale clinical glycosylated proteomics research.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of qualitative and quantitative analysis method of modifying biomarkers in proteomics research biological samples, and particularly relates to a method for applying hydrophilic interaction chromatography coupled with micro-liter flow rate liquid chromatography tandem mass spectrometry to mass spectrometric qualitative and quantitative detection of biomarkers in large-scale clinical biological samples, which is a glycosylated proteomics analysis method based on hydrophilic interaction chromatography coupled with micro-liter flow rate liquid chromatography tandem mass spectrometry. BACKGROUND

[0002] Protein glycosylation is a very common, complex, and diverse post-translational modification of proteins (Reference 1. Moremen K W, Tiemeyer M, Nairn A V. Vertebrate protein glycosylation: diversity, synthesis and function. Nature Reviews Molecular Cell Biology, 2012, 13(7):448–462.). Based on the different ways in which glycans are linked to proteins, protein glycosylation is mainly divided into four types: N-glycosylation, O-glycosylation, C-glycosylation, and glycosylphosphatidylinositol (GPI) anchoring. Among these, N- and O-glycosylation have been the most extensively studied. In N-glycosylation, the glycan is covalently linked to the amino group of the asparagine side chain via N-acetylglucosamine. The glycan has a pentasaccharide core structure, and the glycosylation site has a specific peptide sequence Asn-X-Ser / Thr (X≠Pro). N-glycosylation plays an important role in protein stability, intracellular and intercellular signal transduction, hormone activation or inactivation, and immune regulation. Abnormal expression of protein glycosylation often foreshadows the development of related diseases, such as inflammation, cancer, and genetic diseases (Reference 2. Schjoldager KT, Narimatsu Y, Joshi HJ, Clausen H. Global view of human protein glycosylation pathways and functions. Nat Rev Mol Cell Biol, 2020, 21(12):729–749.). Currently, most approved tumor biomarkers for clinical use are glycoproteins (e.g., alpha-fetoprotein (AFP) for liver cancer, cancer antigen 125 (CA125) for ovarian cancer, carcinoembryonic antigen (CEA) for colon cancer, and prostate-specific antigen (PSA) for prostate cancer) or carbohydrate antigens (e.g., cancer antigen 19-9 (CA19-9) for gastrointestinal and pancreatic cancer) (Reference 3. Wang M, Zhu J, Lubman DM, Gao C. Aberrant glycosylation and cancer biomarker discovery: a promising and thorny journey. Clin Chem Lab Med, 2019, 57(4):407–416.). Therefore, glycoproteomics analysis is very important for early disease screening and pathological research.

[0003] Mass spectrometry-based proteomics provides an excellent analytical tool for the comprehensive analysis of proteins and their modifications (Reference 4. Suttapitugsakul S, Sun F, Wu R. Recent Advances in Glycoproteomic Analysis by Mass Spectrometry. Anal Chem, 2020, 92(1):267–291.). In bottom-up proteomics studies, single proteins or whole proteins from complex biological samples (cells, tissues, body fluids, etc.) are usually digested into peptides by enzymes before qualitative and quantitative analysis by high-performance liquid chromatography-tandem mass spectrometry. However, due to the diversity and high heterogeneity of glycans, comprehensive analysis of protein glycosylation remains a significant challenge. On the one hand, although protein glycosylation is ubiquitous, glycoproteins are less abundant than whole proteins, and the content of intact glycopeptides is even lower, making direct detection by mass spectrometry difficult. Therefore, developing methods for enriching intact glycopeptides is of great importance in order to improve the abundance of intact glycopeptides in complex biological samples. Currently, common glycopeptide enrichment techniques include hydrophilic interaction chromatography, metal affinity chromatography, lectin affinity chromatography, boric acid chemistry, enzymatic chemistry, and acylhydrazine chemistry, etc. (Reference 5. Liu Luyao, Qin Hongqiang, Ye Mingliang. Research progress on new technologies for enrichment and mass spectrometry analysis of intact glycosylated peptides. Chromatography, 2021, 39(10).). Among them, hydrophilic interaction chromatography is a broad-spectrum enrichment method that achieves the separation and enrichment of intact glycopeptides by utilizing the difference in polarity between glycopeptides and non-glycopeptides. It enriches glycopeptide types without bias and preserves the complete structure of glycopeptides.

[0004] Currently, in the experimental procedure for enriching intact glycopeptides using hydrophilic interaction chromatography, due to the lack of commercially available microcolumns or the poor performance of existing commercially available microcolumns, researchers typically fill the enrichment material into self-made microcolumns and perform experimental operations such as equilibration, loading, elution, and eluenting in a centrifugation-assisted microcolumn system. This traditional enrichment procedure is cumbersome, highly susceptible to the influence of experimental personnel, and not conducive to the separation and analysis of large numbers of samples. In 2021, we developed a liquid chromatography-based method for glycopeptide enrichment, which can automate the separation and collection of intact glycopeptides and non-glycopeptides. The method is highly reproducible, easy to operate, and can be run for a long time, making it suitable for the separation and enrichment of intact glycopeptides in large cohorts of samples (Reference 6. Liu L, Zhu B, Fang Z, Zhang N, Qin H, Guo Z, Liang X, Yao Z, Ye M. Automated Intact Glycopeptide Enrichment Method Facilitating Highly Reproducible Analysis of Serum Site-Specific N-Glycoproteome. Anal. Chem., 2021, 93(20): 7473–7480.).

[0005] Nanofluid chromatography (NLC) has been a mainstay of proteomics research for over 20 years, primarily because low flow rates improve the ionization efficiency of peptides in electrospray ionization (ESI), thereby enhancing sensitivity. However, long-term stable operation of NLC requires consideration of factors such as column manufacturing reproducibility, ESI stability, column overload, mass spectrometry saturation, and long loading times due to low flow rates. These factors can limit the reproducibility of qualitative and quantitative peptide analysis, as well as the comprehensiveness, robustness, and throughput of proteomics analysis. A highly stable liquid chromatography-mass spectrometry platform is particularly crucial when analyzing highly complex samples, such as those from tissues and body fluids, or those with a wide dynamic range of protein concentrations. In 2020, Bian et al. developed a microfluidic liquid chromatography-tandem mass spectrometry method for proteomics research. They found that the microfluidic system could operate stably for a long time, greatly improving the stability of proteomics mass spectrometry analysis (Reference 7. Bian Y, Zheng R, Bayer FP, Wong C, Chang YC, Meng C, Zolg DP, Reinecke M, Zecha J, Wiechmann S, Heinzlmeir S, Scherr J, Hemmer B, Baynham M, Gingras AC, Boychenko O, Kuster B. Robust, reproducible and quantitative analysis of thousands of proteomes by micro-flow LC-MS / MS. Nat Commun, 2020, 11(1):157.). Subsequently, we applied this system to the mass spectrometry analysis of intact glycopeptides and found that compared with traditional mass spectrometry analysis systems, its first-order quantification was very stable, and the reproducibility of qualitative and quantitative data was high, which can be effectively applied to the mass spectrometry analysis of glycosylated proteomics of a large number of samples.

[0006] Current research increasingly demonstrates a close correlation between abnormal protein glycosylation levels and human disease states. However, clinical studies often require large sample sizes. Glycosylated proteomics typically involves enriching glycopeptides, leading to cumbersome experimental procedures that hinder large-scale application. While nanofluid chromatography-tandem mass spectrometry (LC-MS / MS), a traditional omics detection method, often experiences system crashes due to pressure buildup after continuous operation with large sample counts, making long-term stable operation difficult. Therefore, this invention provides a highly reproducible method for analyzing glycosylated proteomics in biological samples using a specific chromatographic column at a low flow rate. This method is suitable for long-term stable operation, and its continuous and automated enrichment of N-glycopeptides in large samples holds promise for large-scale clinical glycosylated proteomics studies. The method is simple in principle, easy to operate, and enables qualitative and quantitative analysis of trace amounts of intact glycopeptides in complex biological samples from large cohorts.

[0007] This invention utilizes a chromatographic column of a specific specification to enrich intact glycopeptides using a hydrophilic interaction chromatography system, and then couples this enrichment with a low-flow-rate liquid chromatography-tandem mass spectrometry (LC-MS / MS) system for detecting intact glycopeptides. This enables the study of glycosylated proteomics in large cohorts of biological samples. The experimental procedure of this invention is easy to operate, highly reproducible, and stable, making it suitable for glycosylated proteomics studies of large cohorts of biological samples. This invention is used for the highly reproducible, automated, and long-term stable separation and detection of intact N-linked glycosylated peptides in complex biological samples. Specifically, after obtaining biological samples, peptides are obtained through denaturing enzymatic digestion of proteins. After desalting, the samples are fed into a liquid chromatograph equipped with a hydrophilic interaction chromatography column containing clicked maltose microspheres. The intact glycopeptides in the sample are automatically enriched, and then coupled with a low-flow-rate LC-MS / MS system for detection. Summary of the Invention

[0008] The purpose of this invention is to provide a highly reproducible, automated, long-term stable, and easy-to-operate method for glycosylated proteomics analysis. This invention combines two specific chromatographic columns. First, a liquid chromatograph equipped with a hydrophilic column is used to enrich intact glycopeptides in the enzymatic hydrolysate of biological samples. Then, high-speed liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) at a low flow rate is used for qualitative and quantitative analysis of the intact glycopeptides.

[0009] This patent is the first to integrate a complete glycopeptide enrichment strategy with a low-flow-rate liquid chromatography-tandem mass spectrometry (LC-MS / MS) detection method, and has been successfully applied to long-term glycosylation proteomics research. Compared with traditional glycosylation proteomics analysis methods, this patent utilizes a specific chromatographic column and a complete glycopeptide enrichment strategy based on liquid chromatography to improve the reproducibility of large-scale sample preparation. The low-flow-rate LC-MS / MS detection method improves the stability of long-term sample detection. Overall, it achieves highly reproducible qualitative and quantitative analysis of complete glycopeptides in trace amounts of complex biological samples over a long period. The entire experimental procedure is easy to operate, yields excellent data reproducibility, and is suitable for long-term stable operation, laying the foundation for future large-scale clinical sample glycoproteomics research.

[0010] To achieve the above objectives, the following steps can be taken:

[0011] 1) Take biological samples and reduce and alkylate them in denaturing agents;

[0012] 2) Add buffer solution to the final solution from step 1) and add trypsin to enzymatically digest the protein;

[0013] 3) Acidify the final solution from step 2) to remove salt, collect the desalted eluent, and freeze-dry the sample;

[0014] 4) The sample obtained in step 3) is reconstituted in the enrichment loading solution and submitted to a liquid chromatograph equipped with a hydrophilic interaction column to enrich intact glycopeptides, collect the glycopeptide fraction, and freeze-dry the sample.

[0015] 5) The sample obtained in step 4) is reconstituted in the mass spectrometer loading solution and submitted to a liquid chromatography-tandem mass spectrometer equipped with a C18 column and a flow rate of microliters for separation and detection using the mass spectrometry method.

[0016] 6) Use glycopeptide retrieval software to search for complete glycopeptides in the raw file obtained in step 5), and obtain the qualitative and quantitative results of the complete glycopeptides.

[0017] Supplementary explanation of the plan:

[0018] a. The denaturing agent in step 1) is a 100mM ammonium bicarbonate buffer solution (pH 7.8-8.5) containing 8M urea, the reducing agent has a final concentration of 15-25mM dithiothreitol solution, and the alkylating agent has a final concentration of 30-50mM iodoacetamide solution.

[0019] b. The buffer solution in step 2) is a 50-150 mM ammonium bicarbonate solution (pH 7.8-8.5);

[0020] c. The acidifying reagent in step 3) is a 0.1-1% trifluoroacetic acid solution (by volume).

[0021] d. The enrichment loading solution in step 4) is an acetonitrile solution with a volume concentration of 80-82% (containing 0.1% trifluoroacetic acid by volume);

[0022] e. The mass spectrometry loading solution in step 5) is a formic acid solution with a volume concentration of 0.1-0.3%;

[0023] f. The hydrophilic interaction chromatographic column described in step 4) is packed with clicked maltose microspheres with a particle size of 4-6 μm, and the specifications of the hydrophilic interaction chromatographic column are an inner diameter of 1.9-2.3 mm × a length of 4-6 cm.

[0024] g. The C18 chromatographic column described in step 5) is packed with C18 microspheres with a particle size of 1-3 μm and a column specification of inner diameter 0.8-1.2 mm × length 12-18 cm.

[0025] h. Step 4) The glycopeptide enrichment method is as follows: the system flow rate is set to 180-220 μL / min. After loading the sample, the system is first run with a mobile phase of 80-82% acetonitrile (containing 0.1% trifluoroacetic acid) for 10-12 min. Then, the mobile phase is adjusted to 28-30% acetonitrile (containing 0.1% trifluoroacetic acid) for 2-4 min. The elution curve obtained by a UV detector with a wavelength of 205 or 240 nm is monitored, and the elution peak that appears after 10 min of operation is collected. This elution peak is the glycopeptide of the sample.

[0026] i. Step 5) describes a chromatography-mass spectrometry (GC-MS) method consisting of two parts: separation parameters and detection parameters. The separation parameters are as follows: flow rate set to 50–60 μL / min; mobile phase A is a 0.1–0.3% (v / v) formic acid solution; mobile phase B is an 80–82% (v / v) acetonitrile solution (containing 0.1–0.3% (v / v) formic acid); after sample loading, the mobile phase concentration is increased from 4% B phase to 95% B phase within 54 min, and then the 95% B phase and 4% B phase are maintained in equilibrium, with the overall liquid phase gradient maintained within 60 min. The detection parameters are as follows: the mass spectrometer uses a positive ion detector in DDA mode; the primary spectrum acquisition parameters are a resolution of 70,000 and a maximum allowable injected charge number of 1 × 10⁻⁶. 6 The maximum ion implantation time is 20 ms, and the mass-to-charge ratio range is 350-1800; the secondary spectrum acquisition parameters include a resolution of 35000 and a maximum allowable implantation charge number of 2×10⁻⁶. 5 The maximum ion implantation time is 100ms, the isolation window is 2 mass-to-charge ratios, the step-normalized collision fragmentation energy is 20%-30%-40%, and the dynamic removal time is 15s.

[0027] j. Step 6) The glycopeptide retrieval software is Glyco-Decipher. Data processing uses primary spectrometry for standard-free quantitative analysis and secondary spectrometry for qualitative analysis of glycopeptides.

[0028] The method is simple in principle and easy to operate, and can realize the qualitative and quantitative analysis of complete glycopeptides in a large cohort of complex biological samples.

[0029] Advantages of this invention:

[0030] This method boasts a simple and automated experimental procedure, minimally affected by experimental conditions and personnel. Utilizing a specific chromatographic column, a strategy of enriching intact glycopeptides using a hydrophilic interaction-based liquid chromatography column improves experimental reproducibility. The detection method employing micro-flow rate liquid chromatography-tandem mass spectrometry enhances analytical stability, overcoming issues such as overpressure and overload that arise during long-term operation. This method can be applied to glycosylation proteomics studies of large cohorts of clinical biological samples and holds significant potential for discovering novel disease biomarkers. Attached Figure Description

[0031] Figure 1 This is an experimental flowchart illustrating the method of this patent (a glycoprotein analysis method using hydrophilic chromatography coupled with micro-flow rate chromatography-mass spectrometry) for analyzing intact glycopeptides in biological samples.

[0032] Figure 2 The liquid phase gradient and its chromatographic elution curve are related to the method of this patent. Figure 2 a represents the liquid phase gradient and elution curve of hydrophilic interaction chromatography for enriching intact glycopeptides; Figure 2 b represents the liquid phase gradient and elution curve of intact glycopeptides analyzed by gas chromatography-mass spectrometry at a flow rate of microliters.

[0033] Figure 3 These are typical glycopeptide secondary profiles and intact glycopeptides in the experimental results of this patented method. Figure 3 a represents the secondary spectrum and structure of the complete glycopeptide Hex(5)HexNAc(4)+VVLHPNYSQVDIGLIK; Figure 3 b represents the secondary spectrum and structure of the complete glycopeptide Hex(6)HexNAc(5)NeuAc(3)Fuc(1)+LANLTQGEDQYYLR; the two colored circles in the figure represent the two isomers of Hex, the square represents HexNAc, the rhombus represents NeuAc, and the triangle represents Fuc.

[0034] Figure 4 To analyze the qualitative and quantitative results of intact glycopeptides. Figure 4 a represents the glycopeptide profiles and identification results of intact glycopeptides from three repeated analyses of the plasma glycoproteome; Figure 4 b represents the quantitative results of analyzing intact glycopeptides by comparing the method of this patent (example), Comparative Example-1, and Comparative Example-2.

[0035] Figure 5 To evaluate the effectiveness of this patented method over a long period of time. Figure 5 a represents the offset of the retention time of the standard peptide in the five standard N-glycopeptide samples; Figure 5 b represents the results of examining the glycopeptide spectra and identifying intact glycopeptides during 8 consecutive days of operation. Detailed Implementation

[0036] The present invention will be further illustrated by way of embodiments below, but the present invention is not limited to the scope of the embodiments described herein.

[0037] Example:

[0038] The specific experimental procedure for the separation and detection of glycosylated peptides in human plasma samples using an automated HILIC-μLC-MS / MS method based on hydrophilic interaction chromatography coupled with micro-flow rate is as follows:

[0039] (1) Preparation of plasma standard samples: Collect plasma samples from 200 volunteers (male-female ratio 1:1), and mix 10 μL of each sample together to prepare plasma standard samples.

[0040] (2) Denaturation of plasma proteins: 5 μL of plasma standard sample (protein content of about 300 μg) from step (1) was added to 45 μL of 100 mM ammonium bicarbonate buffer solution (pH 7.8) containing 8 M urea, and then 20 mM dithiothreitol was added. After mixing well, the mixture was placed in a 37°C water bath for 2 h. Then, 40 mM iodoacetamide was added, and after mixing well, the mixture was placed in a 25°C shaker in the dark for 40 min.

[0041] (3) Preparation of plasma protein hydrolysate: Add 180 μL of 100 mM ammonium bicarbonate buffer solution (pH 7.8) to the final solution of step (2), mix well, add 6 μg of trypsin, mix well, and then enzymatically hydrolyze in a 37℃ water bath for 16 h. Add 12 μL of 20% trifluoroacetic acid solution to the solution after the enzymatic hydrolysis, mix well, and then sonicate for 1 min.

[0042] (4) Preparation of plasma protease hydrolysate peptides: Take a desalting column (Waters, Oasis HLB C18, 10 mg), first add 600 μL of methanol solution for activation, then add 600 μL of 0.1% trifluoroacetic acid solution for equilibration, then add the final solution from step (3) for loading, then add 600 μL of 0.1% trifluoroacetic acid solution for elution, and finally add 400 μL of 80% acetonitrile solution (containing 0.1% trifluoroacetic acid) for elution. Collect this eluent, freeze-dry it and store it in a -20℃ freezer for later use. This is the plasma protease hydrolysate peptide. The solution replacement in the above desalting column should wait until the solution added last time has completely flowed through the desalting column before adding a new solution.

[0043] (5) Enrichment of intact N-glycopeptides: The preparation process of click maltose microspheres is as follows: 2g of silane coupling agent (structure (CH3-O)3Si-(CH2)3-Cl) was weighed, and the silane coupling agent, sodium azide, and catalyst sodium iodide were mixed in 2mL of acetonitrile solvent at a molar ratio of 1:3:0.1 and reacted at 130℃ for 24h; then microsphere-type silica gel (particle size 5μm) was added, with 8mmol of silane coupling agent per gram of silica gel, and heated at 160℃ for 18h, followed by heating at 130℃ for another 24h. After filtration, the product was washed successively with dichloromethane, ethanol, water, and acetone to obtain azide-derived silica; 1g of maltose and 10mL of propyneamine were mixed and reacted at 85℃ for 3 hours. After 6 hours, the reaction system was poured into 50 mL of acetonitrile to precipitate alkynyl-derived maltose. The prepared azide-derived silica was then added to a water / ethanol (volume ratio 10 / 1) mixed solvent, with 50 mL of mixed solvent per gram of silica gel. Alkynyl-derived maltose and a catalyst were then added. The catalyst consisted of copper sulfate and sodium ascorbate (molar ratio 1:1), with the catalyst molar amount being 40% of the alkynyl-derived maltose molar amount. The reaction was carried out at 80 °C for 72 hours. The modified alkynyl-derived maltose was bonded to the silica gel surface through click chemistry to obtain click maltose microspheres with a particle size of 5 μm. Subsequently, a hydrophilic interaction chromatographic column with an inner diameter of 2.1 mm and a length of 5 cm was prepared. The plasma protease-digested peptides obtained in step (3) were redissolved in 36 μL of 80% acetonitrile (containing 0.1% trifluoroacetic acid) solution, and the entire solution was submitted to a liquid chromatograph equipped with a hydrophilic column to enrich the intact glycopeptides. The specific process is as follows: The flow rate of the chromatograph was set to 200 μL / min. After loading the protease-digested peptides, the solution was first washed for 11 min in a mobile phase of 80% acetonitrile (containing 0.1% trifluoroacetic acid) solution, and then washed for 2 min in a mobile phase of 30% acetonitrile (containing 0.1% trifluoroacetic acid) solution. The liquid phase gradient and chromatogram are shown below. Figure 2 As shown in figure a, the eluent corresponding to the elution peak at 12-13 min is collected, which is the plasma N-glycopeptide sample. After lyophilization, the eluent is stored at -20℃ for later use.

[0044] (6) Chromatographic and mass spectrometry analysis of intact N-glycopeptides: The plasma standard sample N-glycopeptides prepared in step (5) were reconstituted in 10 μL of 0.1% formic acid solution, and 8 μL of the solution was sent to a Q-Exactive-HF chromatographic and mass spectrometry instrument equipped with a C18 column (the packing material is C18 microspheres with a particle size of 2 μm, and the column specifications are 1 mm inner diameter × 15 cm length, manufactured by Thermo Fisher Scientific, catalog number 164711) for separation and detection. The separation parameters for the chromatography-mass spectrometry (GC-MS) method were as follows: the chromatographic flow rate was set to 60 μL / min; mobile phase A was a 0.1% formic acid solution (v / v); and mobile phase B was an 80% acetonitrile solution (v / v containing 0.1% formic acid). The liquid phase gradients were as follows: maintaining 4% phase B for 0–0.4 min; linearly increasing from 4% phase B to 12% phase B for 0.4–0.6 min; linearly increasing from 12% phase B to 45% phase B for 0.6–54 min; maintaining 95% phase B for 54–56 min; and maintaining 4% phase B for 56–60 min. The overall 60-min liquid phase gradient and chromatogram are shown below. Figure 2 As shown in b. The detection parameters of the chromatographic-mass spectrometry (GC-MS) method are as follows: the mass spectrometer uses a positive ion detector in DDA mode; the first-stage spectral acquisition parameters are a resolution of 70,000 and a maximum allowable injection charge number of 1 × 10⁻⁶. 6 The maximum ion implantation time is 20 ms, and the mass-to-charge ratio range is 350-1800; the secondary spectrum acquisition parameters include a resolution of 35000 and a maximum allowable implantation charge number of 2×10⁻⁶. 5 The parameters were: maximum ion implantation time of 100 ms, isolation window of 2 mass-to-charge ratios, stepwise normalized impact fragmentation energy of 20%-30%-40%, and dynamic exclusion time of 15 s. The obtained raw files were then used to identify and quantify intact glycopeptides using the glycopeptide search software Glyco-Decipher (version 1.0.2).

[0045] (7) Experimental results of the analytical method: Using the methods in steps (2) to (6), the complete N-glycopeptides of a plasma standard sample were analyzed. From 22,259 secondary spectra in the raw file, 3,491 glycopeptide secondary spectra and 1,393 complete glycopeptides were identified and quantified. The experimental results show that the glycopeptide secondary spectra numbered 7573 in the raw file and its corresponding complete glycopeptide are as follows: Figure 3 As shown in figure a, its glycoform is Hex(5)HexNAc(4) (the two colored circles in the figure represent two isomers of Hex, and the squares represent HexNAc), and its peptide amino acid sequence is VVLHPNYSQVDIGLIK; the secondary spectrum of the glycopeptide numbered 10950 in the raw file of the experimental results and its corresponding complete glycopeptide are shown in figure a). Figure 3As shown in b, its glycoform is Hex(6)HexNAc(5)NeuAc(3)Fuc(1) (in the figure, the rhombus represents NeuAc and the triangle represents Fuc), and its peptide amino acid sequence is LANLTQGEDQYYLR; other glycopeptide secondary spectra and complete glycopeptide structures are similar to those shown in b. Figure 3 similar.

[0046] (8) Stability assessment of the analytical method: A plasma standard N-glycopeptide sample was analyzed using the methods in steps (1) to (6), and the experiment was repeated three times. These three standard samples identified 3491 glycopeptide secondary spectra and 1393 intact glycopeptides, 3464 glycopeptide secondary spectra and 1362 intact glycopeptides, and 3494 glycopeptide secondary spectra and 1348 intact glycopeptides, respectively. The relative standard deviation (relative standard deviation = standard deviation / average value * 100%) for the identification of glycopeptides in the secondary spectra was 0.5%, and the relative standard deviation for the identification of intact glycopeptides was 1.7% (e.g., ...). Figure 4 a) Generally, the smaller the relative standard deviation of the identification quantity, the higher the stability of the method. Therefore, the results of this study demonstrate the good identification stability of this method.

[0047] (9) Examination of the quantitative reproducibility of the analytical method: In step (8), a total of 2068 complete glycopeptides were quantified from the three plasma standard N-glycopeptide samples. The relative standard deviation (also known as the coefficient of variation) of the quantified value of each glycopeptide in the three samples was calculated, and the median of the coefficients of variation of all glycopeptides (the middle number in a set of data arranged in ascending order) was calculated using the median() function in Excel and found to be 6.6% (e.g., Figure 4 (See box plot in "Example" in b). Generally, a median coefficient of variation <15% indicates good reproducibility of the method, and the smaller the median coefficient of variation, the better the reproducibility. Therefore, the results of this study demonstrate the excellent quantitative reproducibility of this method.

[0048] (10) Analysis of large cohort samples for long-term stability assessment: Plasma was collected from 200 volunteers in the clinic, with 5 μL collected from each, for a total of 200 5 μL plasma samples. These plasma samples were subjected to protein denaturation and enzymatic digestion, N-glycopeptide enrichment, and mass spectrometry analysis using steps (2) to (6), with a total continuous chromatographic-mass spectrometry analysis time of approximately 10 days. Simultaneously, 5 standard N-glycopeptide plasma samples were prepared according to steps (1) to (6), and one standard plasma sample was analyzed on days 1, 2, 4, 6, and 8 of the 10-day run. Eleven standard peptide fragments (see...) were extracted from these 5 standard samples. Figure 5 As shown in Figure a), the liquid phase retention time reveals that after one week of operation, the retention time of this method hardly shifted (e.g., ...). Figure 5(As shown in a). Each standard sample identified 3494 glycopeptide secondary spectra and 1348 complete glycopeptides, 3396 glycopeptide secondary spectra and 1368 complete glycopeptides, 3295 glycopeptide secondary spectra and 1317 complete glycopeptides, 3350 glycopeptide secondary spectra and 1328 complete glycopeptides, and 3208 glycopeptide secondary spectra and 1301 complete glycopeptides, respectively. The relative standard deviations of the number of glycopeptide secondary spectra identified and the number of complete glycopeptides identified for these five samples were all less than 3%. In summary, the results of this study demonstrate that this method can maintain very stable glycopeptide data reproducibility over long-term operation.

[0049] Comparative Example-1:

[0050] The automated HILIC-nLC-MS / MS method based on hydrophilic interaction chromatography coupled with nanoliter flow rate is used for the separation and detection of glycosylated peptides in human plasma samples. The specific experimental procedure is as follows:

[0051] (11) Analysis of human plasma N-glycopeptides: Samples were prepared according to steps (1) to (5), and the plasma N-glycopeptide sample obtained in step (5) was reconstituted in 20 μL of 0.1% formic acid solution. Then, 4 μL of the entire solution was transferred to a Q-Exactive-HF chromatographic mass spectrometer equipped with a C18 (C18 microspheres with a particle size of 1.9 μm, manufactured by Dr. Maisch ReproSil-Pur 120C18-AQ) column (column specifications: inner diameter 150 μm × length 15 cm) for separation and detection. The separation parameters for the chromatographic mass spectrometry (GC-MS) analysis method are as follows: the chromatographic flow rate is set to 600 nL / min, mobile phase A is a 0.1% formic acid solution (v / v), and mobile phase B is an 80% acetonitrile solution (v / v containing 0.1% formic acid). The liquid phase gradients are as follows: maintain 4% B phase for 0-7 min, linearly increase from 4% B phase to 12% B phase for 7-10 min, linearly increase from 12% B phase to 45% B phase for 10-40 min, maintain 95% B phase for 40-50 min, and maintain 4% B phase for 50-60 min. The detection parameters of the GC-MS analysis method are consistent with those in step (3). The obtained raw files are then used to identify and quantify intact glycopeptides using the glycopeptide retrieval software Glyco-Decipher (version 1.0.2).

[0052] (12) Evaluation and comparison of analytical methods: Compared with the examples, Comparative Example-1 uses a chromatography-mass spectrometry (MS / MS) method based on hydrophilic interaction coupled with nanoliter flow rate. The main comparison focuses on the differences in the separation and detection of glycopeptide samples between MS / MS systems with nanoliter and microliter flow rates. The experiment was repeated three times using the method in step (11). The complete glycopeptides in the experimental results of these three samples include, but are not limited to, those from...Figure 3 The complete glycopeptides shown are as follows. A total of 2417 complete glycopeptides were quantified from these three samples, and the median coefficient of variation for all glycopeptides was 13.4% (e.g., ...). Figure 4 The box plot in "Comparative Example-1" (b) shows a lower coefficient of variation (6.6%), indicating higher reproducibility. Therefore, the micro-flow rate chromatographic-mass spectrometry (MS / MS) method used in this patent is superior in terms of quantitative reproducibility. Since glycoproteomics belongs to modified proteomics, its modified peptides are low in abundance and highly heterogeneous in complex biological samples. Traditional nano-flow rate detection methods cannot achieve particularly stable quantitative reproducibility. However, in this patent's micro-flow rate detection method, high flow rate and large sample volume allow low-abundance modified peptides to be collected by mass spectrometry more frequently, ultimately achieving excellent quantitative reproducibility. Therefore, this patent method is more suitable for research in modified proteomics, such as glycoproteomics.

[0053] Comparative Example-2

[0054] A method for manually enriching N-glycopeptides using a micro-tip chromatographic column, coupled with a nanoliter flow rate chromatography-mass spectrometry (manual HILIC-nLC-MS / MS) method, was used for the separation and detection of glycosylated peptides in human plasma samples. The specific experimental procedure is as follows:

[0055] (13) Enrichment and analysis of N-glycopeptides in human plasma: Weigh 5 mg of hydrophilic interaction chromatography packing material (the packing material is clicked maltose microspheres with a particle size of 5 μm) into a 600 μL tube, add 100 μL of deionized water, sonicate for 1 min, and centrifuge at 10000 g for 3 min; then carefully aspirate and discard the supernatant with a pipette; add 100 μL of 80% acetonitrile (containing 0.1% trifluoroacetic acid) solution and mix well. After incubating in a shaker at 25°C for 1 hour, centrifuge at 10000g for 3 minutes. Carefully aspirate and discard the supernatant using a pipette. Add 100 μL of 80% acetonitrile (containing 0.1% trifluoroacetic acid) solution, mix well, and transfer to a 200 μL pipette tip (the tip should be pre-stuffed with absorbent cotton to form a column head), thus preparing a micro-tip chromatography column. Place the micro-tip column in a small desktop centrifuge and adjust to a suitable speed for centrifugation. Centrifuge 100 μL of solution for about 8 min until the solution level is just above the chromatographic packing material. Then add 100 μL of 80% acetonitrile (containing 0.1% trifluoroacetic acid) solution and centrifuge for later use. Prepare the sample according to steps (2) to (4), and take the plasma protease hydrolysate obtained in step (4), redissolve it in 100 μL of 80% acetonitrile (containing 0.1% trifluoroacetic acid) solution, sonicate for 3 min, and centrifuge at 10000g for 3 min. Then add all the solution to the micro-tip column, centrifuge at an appropriate speed, and after the solution has completely flowed through, add 60 μL, 20 μL and 20 μL of 80% acetonitrile (containing 0.1% trifluoroacetic acid) solution respectively. Then add 100 μL of 30% acetonitrile (containing 0.1% trifluoroacetic acid) solution twice, centrifuge and collect the eluent, which is human plasma N-glycopeptide. The eluent was lyophilized and then reconstituted in 20 μL of 0.1% formic acid solution. 4 μL of the entire solution was then transferred to a Q-Exactive-HF liquid chromatography-mass spectrometry system equipped with a C18 column (150 μm inner diameter × 15 cm length) and used for separation and detection. The separation and detection parameters of the GC-MS analysis method were consistent with those in step (11). The obtained raw files were used to identify and quantify intact glycopeptides using the glycopeptide search software Glyco-Decipher (version 1.0.2).

[0056] (14) Evaluation and comparison of analytical methods: Compared with the examples, Comparative Example-2 uses a manual enrichment method of N-glycopeptides using a micro-tip chromatographic column coupled with nanoliter flow rate chromatography-mass spectrometry (MS / MS). The main comparison focuses on the differences between manual enrichment based on a micro-tip chromatographic column and automated enrichment based on liquid chromatography. The experiment was repeated three times using the method in step (13). The intact glycopeptides in the results of these three samples include, but are not limited to, those from the following samples. Figure 3 The complete glycopeptides shown are as follows. A total of 948 complete glycopeptides were quantified from these three samples, and the median coefficient of variation for all glycopeptides was 17.4% (e.g., ...). Figure 4 The box plot in "Comparative Example-2" (c) shows a lower coefficient of variation of 6.6%, while the corresponding data in the example is 6.6%. This lower coefficient of variation indicates more reproducible quantitative glycopeptide data. Therefore, the glycopeptide enrichment method used in this patent is superior in terms of quantitative reproducibility. In modified proteomics, enrichment methods for low-abundance modified peptides are particularly crucial. Enrichment methods based on micro-tip chromatography columns are widely used but heavily rely on the reproducibility of manual experimental operations. Even so, the reproducibility of manual enrichment methods is not as good as that of automated enrichment methods. At the same time, automated enrichment methods handle large sample sizes with ease. Therefore, this patent method is more suitable for glycoproteomics research that requires enrichment operations.

[0057] Comparative Example-3:

[0058] The following is the experimental procedure for the automated ZIC-HILIC-nLC-MS / MS method, based on a ZIC-HILIC hydrophilic interaction column coupled with nanoliter flow rate, used for the separation and detection of glycosylated peptides in human plasma samples:

[0059] (15) Enrichment and analysis of human plasma N-glycopeptides: Prepare samples according to steps (2) to (4), and take the plasma protease hydrolysis peptide obtained in step (4), redissolve it in 36 μL of 80% acetonitrile (containing 0.01% trifluoroacetic acid) solution, and submit all solutions to a liquid chromatograph equipped with a commercially available ZIC-HILIC hydrophilic interaction column (ZIC-HILIC packing material, packing particle size of 3.5 μm, column specifications of inner diameter 2.1 mm × length 2 cm, manufacturer Merk) and a flow rate of 200 μL / min to enrich intact glycopeptides. The specific process is consistent with the intact glycopeptide enrichment method in step (3). The eluent containing plasma N-glycopeptides was collected, lyophilized, and then reconstituted in 20 μL of 0.1% formic acid solution. 4 μL of the entire solution was transferred to a Q-Exactive-HF chromatography-mass spectrometry (GC-MS) system equipped with a C18 column (1.9 μm particle size, Dr. Maisch ReproSil-Pur 120 C18-AQ) at a flow rate of 600 nL / min for detection. The separation and detection parameters of the GC-MS method were consistent with those in step (11). The obtained raw files were used to identify and quantify intact glycopeptides using the glycopeptide search software Glyco-Decipher (version 1.0.2).

[0060] (16) Evaluation and comparison of analytical methods: Compared with the examples, Comparative Example-3 adopted an automated hydrophilic interaction chromatography-mass spectrometry (MS / MS) method based on ZIC-HILIC packing material, mainly comparing the differences in glycopeptide enrichment effects between click maltose packing material and ZIC-HILIC packing material. A human plasma N-glycopeptide standard sample was analyzed using the method in step (15). The complete glycopeptides in the experimental results of this sample include, but are not limited to, those in the example. Figure 3 The sample showed intact glycopeptides. Only 1215 intact glycopeptides were identified, lower than the results of the previous example (over 1300 intact N-glycopeptides). Therefore, in terms of sensitivity, the hydrophilic column with click maltose packing used in this patented method is superior. For glycoproteomics research, only one commercially available hydrophilic column with ZIC-HILIC packing exists; however, this column is inferior to our click maltose-packed column in terms of enrichment. Therefore, this patented method offers better enrichment and advantages in glycoproteomics research.

[0061] Method evaluation:

[0062] Through the examples and Comparative Example-1, we can see that the micro-flow rate chromatographic-mass spectrometry system has better quantitative reproducibility in separating and detecting intact glycopeptides compared to the nano-flow rate, thanks to the micro-flow rate matched to the C18 column of a specific specification. Through the examples and Comparative Example-2, we can see that the automated enrichment strategy based on liquid chromatography has better quantitative reproducibility than the manual enrichment method based on micro-tip columns, thanks to the system's greater stability due to the hydrophilic interaction column of a specific specification. Through the examples, Comparative Example-1, and Comparative Example-3, we can see that the hydrophilic interaction column with click maltose packing has better identification sensitivity than the hydrophilic interaction column with ZIC-HILIC packing, thanks to the better specificity of click maltose packing for glycopeptides.

[0063] In summary, there are currently no domestic or international literature or patent reports on automated HILIC-μLC-MS / MS based analytical methods using hydrophilic interaction chromatography coupled with micro-flow rates. This patent is also the first to successfully apply this method to glycosylated proteomics research. Compared to traditional manual enrichment methods based on micro-tip chromatographic columns, this patent employs a complete glycopeptide enrichment strategy based on liquid chromatography, improving the reproducibility of large-scale sample preparation. Compared to traditional nano-flow rate chromatographic-mass spectrometry (MS / MS) methods, this patent uses a micro-flow rate MS / MS method, which, through high flow rates and large sample volumes, increases the detection stability of low-abundance modified peptides and improves quantitative reproducibility. Furthermore, through nearly 10 days of continuous analysis of over 200 plasma N-glycopeptide samples, this method maintains very stable glycopeptide identification and data reproducibility during long-term operation. Overall, it achieves highly reproducible qualitative and quantitative analysis of complete glycopeptides in complex biological samples over a long period. The entire experimental procedure is easy to operate, yields excellent data reproducibility, and is suitable for long-term stable operation, laying the foundation for future large-scale clinical sample glycoproteomics research.

Claims

1. A hydrophilic interaction chromatographic coupling method for N-glycosylated proteomics analysis using micro-flow rate chromatography-mass spectrometry, characterized in that: The specific steps are as follows: Take a biological sample, reduce and alkylate it in a denaturing agent; then add buffer solution, add trypsin, and enzymatically digest the protein; after enzymatic digestion, acidify the solution and desalt it; after desalting, lyophilize the enzymatically digested peptides; reconstitute the enzymatically digested peptides in the enrichment loading solution, and submit it to a liquid chromatograph equipped with a hydrophilic interaction column for N-glycopeptide enrichment; monitor the elution curve, collect the N-glycopeptide fraction, and lyophilize the N-glycosylated peptides; reconstitute the N-glycosylated peptides in the mass spectrometry loading solution, and submit it to a liquid chromatography-tandem mass spectrometry system equipped with a C18 column and a microliter flow rate for separation and detection using a chromatography-mass spectrometry method; finally, use N-glycopeptide retrieval software for qualitative and quantitative analysis of the obtained raw file. The specific operation process is as follows: 1) Take biological samples, disperse them in denaturing agent, first add reducing agent and treat at 35~37℃ for 100~120 min, then add alkylating agent and treat at 20~25℃ for 30~40 min; 2) Add 4 to 8 times the volume of denaturant buffer to the final solution in step 1), and add 1 / 50 to 1 / 20 times the amount of trypsin of the biological sample protein. React at 35 to 37°C for 12 to 20 hours to enzymatically digest the protein. 3) Add trifluoroacetic acid to the final solution in step 2) so that the trifluoroacetic acid accounts for 0.1~1% of the total volume of the final solution in step 2). Then load the sample into the desalting column, first add the desalting eluent, then add the desalting elution buffer. Collect and freeze-dry the desalted elution buffer, which is the enzymatically hydrolyzed peptide. 4) The enzymatically hydrolyzed peptide obtained in step 3) is re-dissolved in the enrichment loading solution and submitted to a liquid chromatograph equipped with a hydrophilic interaction column for N-glycopeptide enrichment. The elution curve is monitored, and the N-glycopeptide fraction is collected and freeze-dried to obtain the N-glycosylated peptide. 5) The N-glycosylated peptide obtained in step 4) is reconstituted in the mass spectrometry loading solution and submitted to a liquid chromatography-tandem mass spectrometry system equipped with a C18 column and a flow rate of microliters for separation and detection using the mass spectrometry method. Finally, the raw file obtained is used to obtain the qualitative and quantitative results of the complete N-glycopeptide using the N-glycopeptide search software Glyco-Decipher. Step 4) describes a hydrophilic interaction chromatographic column, the packing material of which is clicked maltose microspheres. The preparation process involves mixing a silane coupling agent, sodium azide, and a catalyst in an acetonitrile solvent at a molar ratio of 1:1.2~3:0.01~0.1, and reacting at 60~130℃ for 8~24 h. The structure of the silane coupling agent is X3Si-(CH2)3-A, where X is -OCH3 or -OCH2CH3, and A is Cl... - or Br - The catalyst is sodium iodide or potassium iodide. Then, 4-6 µm microsphere silica gel is added, with 2-8 mmol of silane coupling agent per gram of silica gel. The mixture is heated at 140-160 °C for 6-18 h, followed by heating at 60-130 °C for 12-24 h. The product is filtered and washed sequentially with dichloromethane, ethanol, water, and acetone to obtain azide-derived silica. Maltose and propargylamine are mixed, with 1-10 mL of propargylamine per gram of maltose. The mixture is reacted at 50-85 °C for 12-36 h. The reaction system is then poured into acetonitrile to precipitate alkynyl-derived maltose. The prepared azide-derived silica is then added to a water / ethanol mixed solvent with a volume ratio of 1 / 10-10 / 1, with 20-50 mL of the mixed solvent per gram of silica gel. mL, then add alkynyl-derived maltose and a catalyst. The catalyst is copper sulfate and sodium ascorbate in a molar ratio of 1:1~1.

2. The molar amount of the catalyst is 1~40% of the molar amount of alkynyl-derived maltose. The reaction is carried out at 20~80℃ for 24~72 h. The modified alkynyl-derived maltose is bonded to the silica gel surface through click chemistry to obtain click maltose microspheres with a particle size of 4~6 µm. Subsequently, a hydrophilic interaction chromatographic column with an inner diameter of 1.9~2.3 mm × a length of 4~6 cm is prepared. Step 4) describes the N-glycopeptide enrichment method as follows: The system flow rate is set to 180–220 µL / min. After sample loading, the mobile phase is first run for 10–12 min with an acetonitrile solution containing 0.1–0.3% trifluoroacetic acid at a volume concentration of 80–82%. Then, the mobile phase is adjusted to an acetonitrile solution containing 0.1–0.3% trifluoroacetic acid at a volume concentration of 28–30%, and the mobile phase is run for 2–4 min. The elution curve is monitored using a UV detector at a wavelength of 205 or 240 nm. The elution peak appearing after 10 min of operation is collected; this elution peak represents the N-glycopeptides in the sample. Step 5) describes a colorimetric-mass spectrometry (GC-MS) analysis method consisting of two parts: separation and detection. The separation parameters were as follows: system flow rate was set to 50–60 µL / min; mobile phase A was a 0.1–0.3% formic acid solution (v / v); mobile phase B was an 80–82% acetonitrile solution containing 0.1–0.3% formic acid (v / v); after sample loading, the mobile phase concentration was increased from 3–5% B phase to 94–96% B phase within 50–60 min, then maintained at 94–96% B phase for 8–12 min, and then maintained at 3–5% B phase for 8–12 min. The detection parameters were as follows: mass spectrometry energy fragmentation used a stepwise normalized collisional fragmentation energy of 20%–30%–40%; data acquisition used a data-dependent acquisition mode; data processing used primary spectrometry for standard-free quantitative analysis and secondary spectrometry for glycopeptide qualitative analysis. Step 5) The C18 chromatographic column is from Thermo Fisher Scientific. Its packing material is C18 microspheres with a particle size of 1~3 µm. The column specifications are an inner diameter of 0.8~1.2 mm × a length of 12~18 cm.

2. The method according to claim 1, characterized in that: Step 2) The buffer solution is a 50-150 mM ammonium bicarbonate solution with a pH of 7.8-8.

5. Step 3) The desalting column is a 10-60 mg Oasis HLB C18 column from Waters Corporation; Step 3) The desalting eluent is a 0.1-1% trifluoroacetic acid solution; Step 3) The desalting elution solution is an 80-82% acetonitrile solution containing 0.1-1% trifluoroacetic acid. Step 4) The enrichment loading solution is an acetonitrile solution containing 0.1-0.3% trifluoroacetic acid and 80-82% acetonitrile. Step 5) The mass spectrometry loading solution is a formic acid solution with a volume concentration of 0.1~0.3%.

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