CN105987945A - Quantitative method for occupancy of N-bond sialylated sugar chains on glycoprotein and application of quantitative method in hepatoma marker screening - Google Patents

Abstract

The invention relates to a method for quantifying the occupancy of N-linked sialylated sugar chains on glycoproteins and its application in screening liver cancer markers. First, take two equal biological samples containing glycoproteins in the same state, and then treat them separately with sodium periodate solution for differential oxidation, and then use the hydrazide chemical method on the protein level to enrich the differentially oxidized samples Then use different stable isotopes to differentially label the glycopeptides on the hydrazide microspheres, then treat them with the peptide glycosidase PNGase F, and finally perform mass spectrometry analysis on the mixed differentially labeled N-glycopeptides to quantify N-linked Occupancy of sialylated sugar chains. This method can be used for proteomic analysis of glycosylation modification, and can simultaneously obtain the identification results of corresponding glycoproteins, glycopeptides and glycosylation sites, especially for the screening of potential biomarkers of human liver cancer (HCC) . This method is simple and efficient.

Description

A method for quantifying the occupancy of N-linked sialylated sugar chains on glycoproteins and its application in screening liver cancer markers

technical field

The invention belongs to the technical field of glycosylation proteomics in the direction of proteomics research, and specifically relates to a method for high-throughput quantification of the occupancy of N-linked sialylated sugar chains on glycoproteins by differential oxidation and labeling based on hydrazide chemistry and its application in Application in the screening of liver cancer markers.

Background technique

Protein glycosylation, as one of the most common and important post-translational modifications, plays an important role in protein folding, conformational stability and activity. Glycosylated proteins (glycoproteins for short) are involved in the molecular recognition of cells and cells, cells and extracellular matrix, apoptosis, protein interaction, immune response and other life processes. Therefore, abnormal changes in glycoproteins are often accompanied by the occurrence and development of many diseases. That is, abnormal glycoproteins in a certain disease can be used as markers of the disease. At present, most of the disease markers used clinically are glycosylated proteins, such as alpha-fetoprotein (AFP) is a marker protein of liver cancer, cancer antigen 125 (cancer antigen 125) is a marker of malignant tumors, and the specific antigen of prostate cancer etc. Therefore, in-depth research on glycoproteins is of great significance.

The N-linked sialylated sugar chain on glycoprotein is a common and very important glycan, and sialic acid is located at the end of the sugar chain. In cells, sialylated sugar chains have many important biological functions, including the interaction between cell molecules, the formation of cell characteristics, and cell metabolism. Studies on this type of glycosylated protein have shown that abnormal sialylation of glycoprotein is closely related to many cancers such as breast cancer, colon cancer, leukemia, pancreatic cancer, etc. Therefore, proteomics experts pay attention to this type of glycoprotein The degree is getting higher and higher. At present, a variety of glycoproteomics methods have been used to identify and quantify sialylated glycoproteins, including specific lectin methods (document 1. Kubota, K. et al. Analysis of glycopeptides using lectin affinity chromatography with MALDI-TOF mass spectrometry.Analytical chemistry 80,3693-3698, doi:10.1021/ac800070d(2008).), titanium dioxide (document 2.Larsen,M.R.,Jensen,S.S.,Jakobsen,L.A.&Heegaard,N.H.Exploring the sialiome using titanium dioxide massa spectrometry.Mol.Cell.Proteomics 6,1778-1787, doi:10.1074/mcp.M700086-MCP200(2007).) and titanium (Ⅳ) metal ion affinity chromatography (document 3.Zhu, J.et al.A simple integrated system for rapid analysis of sialic-acid-containing N-glycopeptides from human serum.Proteomics 13,1306-1313,doi:10.1002/pmic.201200367(2013).), and improved hydrazide chemical method (document 4. Zeng, Y., Ramya, T.N., Dirksen, A., Dawson, P.E. & Paulson, J.C. High-efficiency labeling of sialylated glycoproteins on living cells. Nature methods 6, 207-209, doi: 10.1038/nmeth.1305 (2009). Literature 5 .Nilsson,J.et al.Enrichment of glycopeptides for glycan structure and attachment site identification. Nature methods 6, 809-811, doi: 10.1038/nmeth.1392 (2009). Document 6. Tian, Y., Esteva, F.J., Song, J. & Zhang, H. Altered expression of sialylated glycoproteins in breast cancer using hydrazide chemistry and mass spectrometry. Mol. Cell. Proteomics, doi: 10.1074/mcp. M111.011403 (2012).). The improved hydrazide chemistry method refers to the selective oxidation of sialylated glycoproteins or glycopeptides with a low concentration of sodium periodate solution at low temperature and in a short period of time to achieve their enrichment and analysis. These reported methods directly compare the difference in sialylation at the same glycosylation site between disease samples and normal samples, but there is no study on the occupancy of sialylated sugar chains on glycoproteins in samples of the same state.

The occupancy ratio of sialylated sugar chains can be defined as the molar ratio of sialylated sugar chains to all sugar chains at the same glycosylation site on a protein. Studies have shown that the terminal sialic acid of sugar chains can be selectively oxidized by low-concentration sodium periodate solution, while all kinds of sugar chains can be oxidized by high-concentration sodium periodate solution. The present invention uses different concentrations of sodium periodate solutions to differentially oxidize glycoproteins in the same biological sample for the first time, and develops a method for quantifying the occupancy of N-linked sialylated sugar chains on glycoproteins at the protein level with high throughput.

Contents of the invention

The purpose of the present invention is to provide a simple, accurate and high-throughput method for quantifying the occupancy of N-linked sialylated sugar chains on glycoproteins in complex biological samples. First, take two equal biological samples containing glycoproteins in the same state, and then treat them separately with sodium periodate solution for differential oxidation, and then use the hydrazide chemical method on the protein level to enrich the differentially oxidized samples Then use different stable isotopes to differentially label the glycopeptides on the hydrazide microspheres, then treat them with peptide glycosidase PNGase F, and finally perform mass spectrometry analysis on the mixed differentially labeled N-glycopeptides to quantify N-linked saliva Occupancy of acidified sugar chains.

Specifically include the following steps,

1) Take two equal parts of biological samples containing glycoproteins in the same state, dissolve them in a buffer solution containing 6-8M Urea and 50-100mM NH ₄ HCO ₃ , heat inactivation in boiling water for 5-10min, deactivate with protein The small molecules in the salt column are removed, and the concentration of the whole protein in the buffer solution is 2-10mg/mL;

2) Two parts of processed samples obtained after step 1) are completed, one is oxidized with a sodium periodate solution of 0.5-1mM with a concentration, and the other is oxidized with a sodium periodate solution of 10-15mM with a concentration, and the oxidized The following two samples were replaced with a buffer solution containing 80-100mM NaAC and 130-150mM NaCl with a protein desalting column, and each of the two solutions was added to the two solutions. Hydrazide microspheres, enriched by shaking at room temperature for 10-12h, the volume ratio of the volume occupied by the hydrazide microspheres to the buffer solution replaced in step 2) is 1:5;

3) The hydrazide microspheres obtained after step 2) were washed with 50-100mM NH ₄ HCO ₃ solution for 2 to 3 times to remove non-glycoproteins, and then dispersed in 6-8M Urea and Concentration is 50-100mM NH ₄ HCO ₃ in the buffer solution, with the DDT that is 10-20mM final concentration and the IAA that concentration is 20-40mM each successively after treatment, then with 80-90% ACN (ACN and H ₂ O volume ratio), 50-100mM NH ₄ HCO ₃ solution was washed away 2 to 3 times, and finally dispersed in 50-100mM NH ₄ HCO ₃ buffer solution, the volume occupied by the hydrazide microspheres itself and the added 50 The volume ratio of -100mM NH ₄ HCO ₃ buffer solution is 1:5, and then add trypsin to digest for 12-16h, the mass ratio of the amount of trypsin added to the protein in the sample taken in step 1) is 1:25 ;

4) The microspheres obtained in step 3) are washed successively with 1.0-1.5M NaCl solution, 80-90% ACN (volume ratio of ACN and H ₂ O), 50-100 mM NH ₄ HCO ₃ solution and water to remove non-specific adsorption and Non-glycopeptides, and dispersed in 200-400μL 50-100mM TEAB, respectively, take 8-16μL 4-8wt% CD ₂ O/CH ₂ O (both CD ₂ O and CH ₂ O are 4%-8%) and Add 0.6-1.2M NaBH ₃ CN to the two samples in turn, react at room temperature for 3-4 hours to achieve dimethyl heavy/light labeling of glycopeptides, 4-8% D ₂ O/CH ₂ O and the volume of the microspheres The ratio is 2:25-4:25, the volume ratio of 0.6-1.2M NaBH ₃ CN to the microsphere itself is 2:25-4:25;

5) After washing the hydrazide microspheres obtained in step 4) with water for 2 to 3 times, add 10-20 mM NH ₄ HCO ₃ buffer solution containing 500-1000 Unites PNGase F and shake the enzyme for 10-12 hours to digest the dimethyl The labeled N-glycopeptidase is cut off, and the corresponding light and heavy standard samples are mixed in equal volumes, and the resulting samples are analyzed by MALDI-TOF/TOF mass spectrometer or LC-MS/MS to quantify N-linked sialylation Occupancy ratio of sugar chains; the ratio of the volume of the buffer solution containing PNGase F added to the volume occupied by the hydrazide microspheres itself is 2:1-4:1.

Wherein, the small molecules removed in step 1) are urea, ammonium bicarbonate and other molecules with a molecular weight less than 7000Da in the sample; the pH of the 100mM TEAB in step 4) is 7.5-8.0.

The method for quantifying the occupancy of N-linked sialylated sugar chains on glycoproteins of the present invention can be used for proteomics analysis of glycosylation modifications, and can simultaneously obtain corresponding glycoproteins, glycopeptides, and glycosylation sites. The identification result can especially be used for the screening of potential biomarkers of human liver cancer (HCC).

The differential oxidation and labeling method based on hydrazide chemistry is used for high-throughput quantification of the occupancy of N-linked sialylated sugar chains on glycoproteins, and the glycoprotein enrichment material used is commercial agarose hydrazide microspheres (Bio-Rad, CA, USA).

The differential oxidation and labeling method based on hydrazide chemistry is used for high-throughput quantification of the occupancy of N-linked sialylated sugar chains on glycoproteins, and the quantitative method used is a relative quantitative method. That is, label the glycopeptide enriched after oxidation with 0.5-1mM sodium periodate solution with CD ₂ O, label the glycopeptide enriched after oxidation with 10-15mM sodium periodate solution with CH ₂ O, and mix the two equimolar amounts Perform mass spectrometry.

Advantages of the present invention:

The method of the invention has obvious advantages: simplicity, high efficiency and high throughput. The hydrazide chemical method for enriching glycopeptides has been widely used in the analysis of glycoproteomics. It has the advantages of easy-to-obtain materials, easy operation, high enrichment specificity, and high efficiency. The experimental process of the present invention uses the hydrazide chemical method at the protein level, so that the entire operation process of one sample can be completed in one sample tube, which greatly simplifies the operation and saves time. For the first time, the present invention utilizes sodium periodate solutions of different concentrations in the hydrazide chemical method to differentially oxidize sugar chains and all types of sugar chains with sialic acid at the end of the same glycoprotein, and then utilizes light and heavy isotope labeling and high-resolution RPLC -MS/MS analysis, so as to achieve efficient and high-throughput quantification of the occupancy of N-linked sialic acid sugar chains in the analyzed samples.

Description of drawings

Below in conjunction with accompanying drawing and embodiment the present invention is described in further detail:

Fig. 1 is an experimental flowchart of the high-throughput quantification of the occupancy of N-linked sialylated sugar chains on glycoproteins by the differential oxidation and labeling method based on hydrazide chemistry.

Figure 2A shows that the differential oxidation and labeling method based on hydrazide chemistry was used to specifically enrich and quantify the occupancy of N-linked sialylated sugar chains on human serum transferrin (Transferrin from human blood plasma, HT). The MALDT-TOF mass spectrum of the ratio; Figure 2B is the differential oxidation and labeling method based on hydrazide chemistry for specific enrichment and quantification of standard glycoproteins—partially desialylated human serum transferrin (Asialo transferrin from human MALDT-TOF mass spectrum of N-linked sialylated sugar chain occupancy on blood plasma (AHT).

Figure 3A is the distribution of the Log ₂ value of the occupancy of N-linked sialylated sugar chains on glycoproteins quantified by the differential oxidation and labeling method based on hydrazide chemistry in human liver cancer tissue (HCC) and normal liver tissue (Normal) Figures; Figure 3B is a graph of the number of N-linked glycosylation sites on glycoproteins that are simultaneously present in human liver cancer tissue (HCC) and normal liver tissue (Normal) quantified by the differential oxidation and labeling method based on hydrazide chemistry.

Figure 4 is the ratio of the occupancy ratio of N-linked sialylated sugar chains on glycoproteins that are simultaneously present in human liver cancer tissue (HCC) and normal liver tissue (Normal) quantified by the differential oxidation and labeling method based on hydrazide chemistry Distribution plot of Log ₂ values.

detailed description

Materials and Reagents:

Human serum transferrin (Transferrin from human blood plasma, HT), sodium periodate (sodium periodate), dithiothreitol (1,4-dithiothreitol, DTT), iodoacetamide (iodacetamide, IAA), pancreatic Protease (trypsin), 2,5-dihydroxyl benzoic acid (2,5-dihydroxyl benzoic acid, DHB) and triethylammonium bicarbonate (triethylammonium bicarbonate buffer, TEAB) buffer were purchased from Sigma (IL, U.S.A.) . Protein desalting columns (Zeba Spin desalting columns) were purchased from Thermo Scientific (IL, U.S.A.). Sepharose hydrazide microspheres (hydrazide sepharose resin) were purchased from Bio-Rad (CA, U.S.A.). Peptide-N-glycosidase F, PNGase F and neuraminidase were purchased from New England Biolabs (MA, U.S.A.). Experimental water was purified by Milli-Q water treatment system purchased from Millipore Corporation (MA, U.S.A.). All other reagents were of analytical grade or higher.

Example 1 Occupancy of N-linked sialylated sugar chains on quantitative standard glycoproteins

Human serum transferrin (HT) is a simple and easy-to-obtain glycoprotein, and the sugar chain ends on its two N-glycosylation sites are all sialic acid (document 7.Satomi, Y., Shimonishi, Y .,Hase,T.&Takao,T.Site-specific carbohydrate profiling of human transferrin by nano-flow liquid chromatography/electrospray ionization mass spectrometry.Rapid communications in mass spectrometry:RCM18,2983-2988,doi:10.1002/rcm.1718( 2004).). The terminal sialic acid can be partially removed by desialidase to obtain desialylated transferrin (AHT) (Reference 6). The experimental flow for quantifying the occupancy of N-linked sialylated sugar chains on the two standard glycoproteins by using the method of the present invention is shown in FIG. 1 .

1. Take two 1mg portions of each of the two standard glycoproteins, dissolve them in 100μL buffer containing 8M Urea and 100mM NH ₄ HCO ₃ (pH 8.2), inactivate them by heating in boiling water for 10min, and remove them with a protein desalting column Small molecule. The next experiment is to operate the four samples separately and in parallel.

2. One part of HT and AHT was oxidized with 1 mM sodium periodate, and the other part was oxidized with 10 mM sodium periodate. After each of the four samples was replaced with a buffer solution (pH 5.5) containing 100mM NaAc and 150mM NaCl with a protein desalting column, each of them was added with 100 μL of hydrazide microspheres (the microspheres themselves occupied volume), enriched overnight by shaking at room temperature.

3. The hydrazide microspheres were washed three times with 500 μL of 100 mM NH ₄ HCO ₃ (pH 8.2) to remove non-glycoproteins, and then dispersed in 500 μL of buffer containing 8M Urea and 100 mM NH ₄ HCO ₃ (pH 8.2). After treatment with a final concentration of 10mM DTT and 20mM IAA, wash with 500μL 80% ACN and 100mM NH ₄ HCO ₃ for three times, and finally disperse them in 500μL 100mM NH ₄ HCO ₃ , add 40μg trypsin to digest overnight .

4. Wash non-specific adsorption and non-glycopeptides with 500 μL 1.5M NaCl, 80% ACN, 100 mM NH ₄ HCO ₃ solution and water in sequence, and disperse in 400 μL 100 mM TEAB (pH 8.0) respectively. 16 μL of 4% CD ₂ O/CH ₂ O and 16 μL of 0.6M NaBH ₃ CN were respectively added to the sample oxidized by 1 mM/10 mM sodium periodate in sequence, and reacted at room temperature for 3 h to achieve dimethyl heavy/light labeling.

5. After washing the hydrazide microspheres with water three times, add 200 μL of 10 mM NH ₄ HCO ₃ buffer solution containing 500 Unites PNGase F and shake overnight to digest the dimethyl-labeled N-glycopeptidase. Mix the light standard and heavy standard samples corresponding to HT and AHT in equal volumes, and the resulting two samples are HT-10mM_L-and-1mM_H and AHT-10mM_L-and-1mM_H. Analyzed by MALDI-TOF/TOF mass spectrometer respectively.

Analysis results:

As shown in Figures 2A and 2B, the lightly and heavily labeled glycopeptides in HT were detected with similar peak intensities, while the peak intensities of the heavily labeled glycopeptides corresponding to the two N-linked glycosylation sites in AHT were significantly lower than Peak intensities of lightly labeled glycopeptides. Figure 2A shows that the occupancy rate of N-linked sialylated sugar chains on the two glycosylation sites of HT is close to 100%, which is consistent with that reported in the literature (Document 7). Figure 2B shows that after the N-linked sialylated sugar chain is desialic acid, the peak intensity of the corresponding glycopeptide is also reduced. Therefore, the occupancy of N-linked sialylated sugar chains on the corresponding glycosylation sites of the two glycoproteins can be calculated from the peak intensity of the heavy-labeled glycopeptide compared to the peak intensity of the upper light-labeled glycopeptide (H/L) ,As shown in Table 1. Obviously, the method designed in the present invention can simply and efficiently quantify the occupancy of N-linked sialylated sugar chains on glycoproteins.

Table 1. Occupancy of N-linked sialylated sugar chains on HT and AHT quantified by the differential oxidation and labeling method based on hydrazide chemistry

Example 2

We used this method to quantify the occupancy of N-linked sialylated sugar chains on glycoproteins in the liver tissues of liver cancer patients and normal people, and compared the N-linked sialylated sugar chains on glycoproteins in the two states Changes in the occupancy rate of HCC, thereby discovering potential biomarkers in human liver cancer.

The human liver tissue samples used in this experiment were provided by the Second Affiliated Hospital of Dalian Medical University (Dalian, China), which are non-cancerous tissues around the liver (≥ 2 cm) of hepatocellular carcinoma (HCC). The normal tissue part (Normal) had been pathologically sliced, and the acquisition and use of the samples were completely legal and in compliance with the relevant regulations of the ethics committee of the hospital. The processing procedure of the protein solution extracted from the liver tissue is the same as that of the standard protein, as shown in Figure 1. Finally, the occupancy rate of 496 site-specific N-linked sialylated sugar chains on 334 glycoproteins was quantified from liver cancer tissues based on the quantification in two or more mass spectrometry repetitions; The occupancy of 632 site-specific N-linked sialylated sugar chains on 394 glycoproteins were quantified in liver tissue. The distribution of the Log ₂ values of the occupancy of N-linked sialylated sugar chains of the two samples is shown in Fig. 3A. It can be seen that the occupancy rate of N-linked sialylated sugar chains in normal liver tissue is generally higher than that in cancer tissue. It can be speculated that the occupancy of N-linked sialylated sugar chains on some specific glycoproteins changed significantly in the two samples. As shown in Figure 3B, 284 N-linked glycosylation sites were present in both samples. We calculated the ratio of the occupancy of N-linked sialylated sugar chains on these 284 sites, and Figure 4 shows the distribution of the Log ₂ value of this ratio. Significant differences were found in the occupancy of sialylated sugar chains on 76 common N-glycosylation sites in the two samples, among which the occupancy of 40 N-linked sialylated sugar chains on 37 glycoproteins was significantly down-regulated , the occupancy of 36 N-linked sialylated sugar chains on 31 glycoproteins was significantly up-regulated. These glycoproteins can be used to further screen for markers of human liver cancer.

in conclusion

Apparently, the method developed by the present invention can quantify the occupancy of N-linked sialylated sugar chains on glycoproteins in complex biological samples in a simple, efficient and high-throughput manner, providing a method for screening disease markers that is not affected by protein abundance. The new idea will play an important role in the study of disease markers.

Claims (6)

1. the method that on quantitative glycoprotein, N-connects sialylated sugar chain occupation rate, its feature It is: first take the biological sample equivalent two parts containing glycoprotein under same state, then with height It is processed to carry out differential oxidation by sodium iodide solution respectively, utilizes the acyl on albumen level afterwards The glycoprotein in sample after hydrazine chemical method enrichment differential oxidation, the different stable isotope of recycling Glycopeptide on difference labelling hydrazides microsphere, then with peptide glycosidase PNGase F process, finally The N-glycopeptide of mix variance labelling is carried out mass spectral analysis, connects sialylated sugar chain with quantitative N- Occupation rate.

On quantitative glycoprotein the most according to claim 1, N-connects sialylated sugar chain and occupies The method of rate, it is characterised in that: specifically comprise the steps of,

1) take the biological sample equivalent two parts containing glycoprotein under same state, be dissolved in respectively Containing 6-8M Urea and 50-100mM NH₄HCO₃Buffer solution in, boiling water is heated and inactivated 5-10min, removes little molecule therein with albumen desalination post, and whole protein is in buffer solution Concentration is 2-10mg/mL；

2) step 1) terminate after two parts of treated samples obtaining, portion concentration is The sodium periodate solution oxidation of 0.5-1mM, another part is the periodic acid of 10-15mM by concentration Sodium solution aoxidizes, and two samples after oxidation are respectively replaced into albumen desalination post and containing concentration are The buffer solution of the NaCl of 80-100mM NaAC and 130-150mM, in two solution Each addition washes from the hydrazides microsphere crossing 2～3 times, room temperature concussion enrichment with this buffer solution in advance 10-12h, the shared volume of hydrazides microsphere self and step 2) body of buffer solution replaced Long-pending ratio is 1:5；

3) step 2) terminate after the hydrazides microsphere concentration that obtains be 50-100mM's NH₄HCO₃Solution is washed from 2～3 times to remove non-glycoprotein, is respectively dispersed in and containing concentration is The Urea of 6-8M and concentration are the NH of 50-100mM₄HCO₃Buffer in, with eventually the denseest After DDT that degree is 10-20mM and IAA that concentration is 20-40mM processes the most successively, The most successively with 80-90%ACN (ACN and H₂The volume ratio of O), 50-100mM NH₄HCO₃ Solution is respectively washed from 2～3 times, and last Monodispersed is at 50-100mM NH₄HCO₃In, hydrazides is micro- The 50-100mM NH of the shared volume of ball self and addition₄HCO₃The volume ratio of buffer is 1:5, respectively add trypsin digestion 12-16h, the tryptic consumption of addition and step 1) in the sample taken, the mass ratio of protein is 1:25；

4) step 3) microsphere that obtains is each successively with 1.0-1.5M NaCl solution, 80-90%ACN (ACN and H₂The volume ratio of O), 50-100mM NH₄HCO₃Non-spy is gone in solution and washing Opposite sex absorption and non-glycopeptide, and be dispersed in respectively in 200-400 μ L 50-100mM TEAB, Take 8-16 μ L 4-8wt%CD respectively₂O/CH₂O(CD₂O and CH₂O is 4%-8%) With 0.6-1.2M NaBH₃CN is respectively added sequentially in two kinds of samples, room temperature reaction 3-4h with Realize the diformazan basic weight/light labelling of glycopeptide, 4-8%D₂O/CH₂O with microsphere own vol ratio is 2:25-4:25,0.6-1.2M NaBH₃CN is 2:25-4:25 with the volume ratio of microsphere self；

5) washing with water from step 4) after the hydrazides microsphere 2 that obtains～3 times, each add containing 500 The 10-20mM NH of-1000Unites PNGase F₄HCO₃Buffer concussion enzyme action 10-12 H, scales off the N-glycopeptidase of dimethyl labelling, by corresponding light mark and weight target sample with Equal-volume mixes, the sample of gained, with MALDI-TOF/TOF mass spectrometer or LC-MS/MS analyzes, and connects the occupation rate of sialylated sugar chain with quantitative N-；Wherein add Shared by the volume of the buffer solution containing PNGase F and hydrazides microsphere self, the ratio of volume is 2:1-4:1。

On quantitative glycoprotein the most according to claim 2, N-connects sialylated sugar chain and occupies The method of rate, it is characterised in that: step 1) described in the little molecule of removing be carbamide, carbonic acid Other molecular weight molecule less than 7000Da in hydrogen ammonium and sample.

On quantitative glycoprotein the most according to claim 2, N-connects accounting for of sialylated sugar chain The method having rate, it is characterised in that: step 4) described in the pH of 100mM TEAB be 7.5-8.0。

5. on the quantitative glycoprotein described in a claim 1, N-connects sialylated sugar chain and occupies The application of the method for rate, it is characterised in that: the method may be used for glycosylation modified protein Group credit analysis, can obtain the qualification result of corresponding glycoprotein, glycopeptide and glycosylation site simultaneously.

On quantitative glycoprotein the most according to claim 5, N-connects sialylated sugar chain and occupies The application of the method for rate, it is characterised in that: the method is particularly useful for people's hepatocarcinoma (HCC) The screening of potential source biomolecule mark.