Method for enriching Afamin complete N-glycopeptide in serum and detecting Afamin complete N-glycopeptide in serum by LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry)

Abstract

The invention discloses a method for detecting serum Afamin complete N-glycopeptide through enrichment and LC-MS / MS. The method for enriching the serum Afamin N-glycopeptide comprises the step of simultaneously enriching target glycoprotein / glycopeptide on the protein level and the peptide fragment level in sequence. According to the method, interference of non-glycosylated high-abundance protein is removed, meanwhile, the influence of non-glycosylated high-abundance peptide fragments in glycoprotein is removed, meanwhile, the sample obtained through enrichment with the method can be used for detecting complete glycopeptide of target protein, namely, by detecting the glycopeptide obtained through enrichment with the method, the detection accuracy is high, and the detection sensitivity is high. According to the present invention, the glycosylation modification site of the target protein can be known, the sugar chain composition and the structure of the glycosylation modification amino acid site can be known, the cost is low, the operation is simple and easy, and the foundation is laid for the analysis of the Afamin complete N-glycopeptide.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, in particular to a method for enriching and detecting serum Afamin complete N-glycopeptide by LC-MS / MS. Background Art

[0002] Glycosylation is one of the most important post-translational modifications of proteins. About 50% of proteins in the human body are glycosylated. Protein glycosylation not only has the functions of receptor interaction, immune response and protein secretion and transportation, but also affects the solubility, stability and folding of proteins, and affects the spatial structure and function of proteins. Glycosylation changes with changes in lifestyle or physiological conditions. Glycosylation has a significant impact on a variety of diseases such as cancer, diabetes, and cardiovascular disease through protein function.

[0003] Afamin is a multifunctional glycoprotein that is closely related to the onset of pregnancy complications such as obesity, diabetes, ovarian cancer, and eclampsia. Afamin is an 87 kD glycoprotein with a sugar content of 15% and 5 predicted glycosylation modification sites. Early research data showed that more than 90% of Afamin has 2-6 and 2-3 sialic acid double antennae structures. The glycosylation modification of Afamin may play a major role in the function of Afamin. However, the functional research on the sugar chain structure of Afamin is not thorough. There are studies, but the current research on the glycosylation modification of serum Afamin is seriously insufficient.

[0004] Serum samples are complex and diverse in composition, with limited glycoprotein content and a low glycosylation modification ratio. They are easily inhibited and interfered by other non-glycoproteins or non-glycopeptides during detection, and glycoproteins or glycopeptides need to be enriched before analysis and detection. At present, the glycoprotein / glycopeptide enrichment methods generally include lectin method, boric acid method, hydrazine chemical method, Hilic hydrophilic interaction method, and various synthetic material enrichment methods. On the one hand, the boric acid method and hydrazine chemical method have complicated operation steps, and complete glycopeptides cannot be detected, and the cost of Hilic chromatographic columns is relatively high. On the other hand, at present, only glycoproteins or only glycopeptides are generally enriched, and glycopeptides are only enriched at the peptide level. High-abundance non-target protein peptides will interfere with and inhibit the detection of target protein glycopeptides; enrichment is only performed at the protein level. First, the cost of multiple affinity fillers for removing high-abundance serum proteins is high. Secondly, due to the low glycosylation modification ratio, non-glycopeptides of the target protein will interfere with and inhibit glycopeptide analysis and detection. Summary of the invention

[0005] The purpose of this method is to develop a more effective and low-cost method for enriching serum Afamin N-glycopeptide based on the characteristics of the target protein Afamin.

[0006] The present invention provides a method for enriching serum Afamin N-glycopeptide, comprising the steps of simultaneously enriching target glycoprotein / glycopeptide at protein and peptide levels in sequence.

[0007] Wherein, the step of enriching the target glycoprotein / glycopeptide at the protein level comprises: 1) Removing IgG from serum proteins in the serum sample to be tested to obtain a processed serum protein sample; 2) The serum protein sample obtained in step 1 is further processed to obtain a high mannose type Afamin protein sample.

[0008] Wherein, the specific method for removing IgG from serum proteins in the serum sample to be tested in step 1) is to use Protein G to remove IgG from serum proteins.

[0009] The method for further processing the serum protein sample in step 2) is as follows: dilute the serum protein sample and add it to the Con A affinity column, collect the flow-through and recover the column binding buffer to obtain the flow-through protein sample; elute the glycoprotein bound to Con A using an eluent, collect the eluted protein (the elutant) to obtain the eluted protein sample; concentrate the flow-through protein sample and the eluted protein sample to equal volumes, combine them, and obtain a high mannose type Afamin protein sample.

[0010] Wherein, the step of enriching the target glycoprotein / glycopeptide at the polypeptide level comprises: 3) The high mannose-type Afamin protein sample obtained in step 2 is enzymatically digested to obtain a polypeptide mixture.

[0011] 4) The peptide mixture is enriched for N-linked glycopeptides of Afamin using an organic solvent method to obtain a serum protein sample that has been enriched.

[0012] The enzymatic cleavage method in step 3) is as follows: the high mannose type Afamin protein sample is added to dithiothreitol and incubated at 37°C for 1 hour, iodoacetamide is added and incubated in the dark for 45 minutes, and the m 蛋白质 :m 酶 =1:40, trypsin was added and incubated at 37°C for 16 hours, the polypeptide was recovered, and vacuum-frozen into dry powder to obtain a polypeptide mixture.

[0013] The enrichment method in step 4) is as follows: resuspend the polypeptide sample, add 10 times the volume of 1% formic acid / acetone (volume ratio), vortex mix, incubate, and centrifuge to recover the precipitate; spin dry the supernatant using a vacuum centrifugal concentrator; and dissolve the precipitate using a mixture of acetonitrile, water and formic acid to obtain a serum polypeptide sample that has been enriched.

[0014] The present invention also provides a method for detecting serum Afamin complete N-glycopeptide by LC-MS / MS, comprising the step of using the enriched serum protein sample obtained by the above method to perform detection.

[0015] The method comprises the steps of performing LC-MS / MS analysis using an Easy-nLC 1200 liquid chromatograph and an Orbitrap Fusion mass spectrometer.

[0016] The mobile phase A was 0.1% FA / H2O, and the mobile phase B was 80% acetonitrile (ACN) / 0.1% FA. The pump flow rate was 2 μL / min, and the sample was loaded onto the analytical column, and the separation flow rate was 300 nL / min.

[0017] Among them, the liquid phase elution program is: 0.0 ~ 3 min: 3% B ~ 8% B; 3 ~ 95 min: 8% B ~ 28% B; 95 ~ 110min: 28% B ~ 32% B; 110 ~ 115 min: 32% B ~ 95% B; 115 ~ 120 min: 95% B; 120 ~ 120.1 min: 95% B ~ 3% B.

[0018] Among them, the DDA-MS method is as follows: spray voltage: 1900V, ion transfer tube temperature: 320℃, mass spectrometry scanning range (m / z) is 375~1500, MS1 resolution 60,000, maximum injection time 35 ms, AGC Target: 4e5; RF lens: 60%; precursor ion charge state: 2-7; dynamic exclusion cycle: 60s; MS2 resolution 30,000, ion fragmentation mode: HCD; maximum injection time 54 ms, AGC Target: 5e4.

[0019] Compared with the traditional method of single enrichment at the protein level or the polypeptide level, the method of the present invention is characterized in that the target glycoprotein / glycopeptide is enriched at the protein and peptide levels in sequence. This method not only removes the interference of non-glycosylated high-abundance proteins, but also removes the influence of non-glycosylated high-abundance peptides in glycoproteins. At the same time, the enriched samples of this method can be used to detect the complete glycopeptides of the target protein, that is, by detecting the glycopeptides enriched by this method, not only the glycosylation modification sites of the target protein can be understood, but also the sugar chain composition and structure of the glycosylated modified amino acid sites can be understood. In addition, this series of methods has low cost and is simple and easy to operate, laying the foundation for the analysis of the complete N-glycopeptide of Afamin. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1This is the Western Blot analysis result of the serum protein Afamin enriched by ConA. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0022] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0023] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0024] Example 1 The present invention provides a method for simultaneously enriching target glycoproteins / glycopeptides at the protein and peptide levels, effectively enriching serum Afamin N-glycopeptides, and performing LC-MS / MS detection, which specifically comprises the following steps: Step 1: Protein G removes IgG from serum proteins Specific implementation steps: Take the serum sample out of the -80 ℃ refrigerator, thaw at 4-8 ℃, and dilute with Binding Buffer (0.15 M NaCl / 10 mM sodium phosphate / 10 mM Na2EDTA, PH 7.0); Balance the filler: Take out the Protein G filler and place it in a 2 ml EP tube, add 1 ml Binding Buffer (repeat 3 times); Take 1 ml of the diluted serum sample and add it to the filler, shake it at room temperature for 1 h; centrifuge at 3000 rpm, 4 ℃ for 1 min, let it stand for 5 min, and recover the supernatant; add 1 ml Binding Buffer, centrifuge at 3000 rpm, 4 ℃ for 1 min, let it stand for 5 min, recover the supernatant, and repeat this step 5 times; Elute IgG: Add 1 ml Elution Buffer (0.5 M ammonium acetate, mix at RT for 10 min, centrifuge at 3000 rpm, 4 ℃ for 1 min, let it stand for 5 min, and recover the supernatant; repeat this step 5 times; Use Binding The filler was washed with buffer, balanced to pH 7.0, and stored in a refrigerator at 4°C. The serum protein sample was obtained after IgG removal.

[0025] Step 2: High-mannose Afamin Sample Preparation Specific implementation steps: Con A SepharoseTM 4B was loaded into a centrifugal spin column. The serum protein sample was diluted with binding buffer (20 mmol / L Tris-HCl / 0.5 mol / L NaCl / 1 mmol / L MnCl2 / 1mmol / L CaCl2) and filtered through a 0.45 μm filter membrane. The treated sample was added to the equilibrated Con A affinity column and shaken overnight at 4°C. The flow-through was collected by centrifugation at 1,000 g. The column was washed 5 times with 10 times the sample volume of binding buffer and the binding buffer for washing the column was recovered and combined with the flow-through. The column was washed 5 times with 10 times the sample volume of 0.3 mol / L methyl-α-D-pyranoglucoside (methyl-α-D-glucopyranoside) / 20 mmol / L Tris-HCl / 0.5 mol / LNaCl (pH 7.4) to elute the glycoprotein bound to Con A and collect the eluted protein (the elutant). The flow-through protein sample and the eluted protein sample were concentrated to equal volumes using a kD ultrafiltration tube to obtain a high-mannose Afamin protein sample.

[0026] Western Blot analysis was performed on total serum protein of equal mass, protein flow-through of Con A affinity chromatography, and eluted protein. The results are shown in Figure 1. Afamin was detected in both the original serum protein and eluted protein samples, but not in the flow-through protein sample. That is, all Afamin in serum was bound by Con A. Based on the specific binding property of Con A to high-mannose glycoproteins, it was further concluded that serum Afamin was glycosylated with high mannose and could be enriched by ConA.

[0027] Step 3: FASP (filter aided sample preparation) enzymatic digestion with trypsin Specific implementation steps: add 10 mM dithiothreitol to the protein sample and incubate at 37 °C for 1 h, incubate at room temperature in the dark with 40 mM iodoacetamide for 15 min, add trypsin and incubate at 37 °C overnight, recover, and vacuum freeze into dry powder to obtain a polypeptide mixture.

[0028] Step 4: Enrichment of N-linked glycopeptides of Afamin by organic solvent method Specific implementation steps: resuspend the peptide sample, add 10 times the volume of 1% formic acid / acetone, vortex mix, incubate at -20℃ for 18 hours, centrifuge at 15,000 g to recover the precipitate and enrich the glycopeptide, and dissolve the precipitate with 2% acetonitrile / 98% H2O / 0.1% formic acid to obtain the enriched serum high mannose glycopeptide sample.

[0029] Step 5: LC / MS / MS separation and analysis Specific implementation steps: LC-MS / MS analysis was performed using an Easy-nLC 1200 liquid chromatograph and an Orbitrap Fusion mass spectrometer, with mobile phase A being 0.1% FA / H2O and mobile phase B being 80% acetonitrile (ACN) / 0.1% FA. The pump flow rate was 2 μL / min, and the sample was loaded onto the analytical column, with a separation flow rate of 300 nL / min. Liquid phase elution program: 0.0 ~ 3 min: 3% B ~ 8% B; 3 ~ 95 min: 8% B ~ 28% B; 95 ~ 110 min: 28% B ~ 32% B; 110 ~ 115 min: 32% B ~ 95% B; 115 ~ 120 min: 95% B; 120 ~ 120.1 min: 95% B ~ 3% B.

[0030] DDA mass spectrometry parameters: mass spectrometry scanning range (m / z) was 300-1,500, MS1 resolution was 60,000, maximum injection time was 50 ms, AGC Target: 4e5; MS2 resolution was 15,000, maximum injection time was 100 ms, AGC Target: 5e4.

[0031] Step 6: Database Search Specific implementation steps: retrieval software: pGlyco 3.0, glycosylation modification type: N-linked, glycoside database: pGlyco.gdb, enzyme cutting type: Trypsin_KR-C, missed cutting sites: 2, parent ion error: 10ppm, daughter ion error: 20ppm, fixed modification: Carbamidomethyl[C], variable modification: Oxidation[M], Deamidated[N].

[0032] Example 2 10μL and 2μL of the same serum were removed respectively; the 10μL serum sample was treated according to the conventional method of removing serum (TOP14) high-abundance proteins + trypsin digestion in the prior art; the 2μL serum sample was treated according to the method of the present invention (trypsin digestion + organic solvent method to enrich serum glycopeptides). From the test results in Table 1, it can be seen that in the 10μL sample without organic solvent enrichment after trypsin digestion, 568 reliable glycopeptides (false discovery rate (FDR) < 0.01)) were detected; in another set of data, 2μL serum samples were digested by trypsin and then enriched by organic solvent method for serum glycopeptides, and 2231 effective glycopeptides were detected. This shows that the organic solvent method significantly improves the detection rate of N-glycopeptides.

[0033] Table 1. N-glycopeptide enrichment effect

[0034] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A method for enriching serum Afamin N-glycopeptide, characterized in that: The method includes the steps of simultaneously enriching the target glycoprotein / glycopeptide at the protein and peptide levels.

2. The method according to claim 1, characterized in that The step of enriching the target glycoprotein / glycopeptide at the protein level comprises: 1) Removing IgG from serum proteins in the serum sample to be tested to obtain a processed serum protein sample; 2) The serum protein sample obtained in step 1 is further processed to obtain a high mannose type Afamin protein sample.

3. The method according to claim 2, characterized in that The specific method for removing IgG from serum proteins in the serum sample to be tested in step 1) is to use Protein G to remove IgG from serum proteins.

4. The method according to claim 2, characterized in that: The method for further processing the serum protein sample in step 2) is as follows: dilute the serum protein sample and add it to the Con A affinity column, collect the flow-through liquid and recover the column binding buffer to obtain the flow-through protein sample; elute the glycoprotein bound to Con A with the eluent, collect the eluted protein (the elutant) to obtain the eluted protein sample; concentrate the flow-through protein sample and the eluted protein sample to equal volumes, combine them, and obtain the high mannose type Afamin protein sample.

5. The method according to claim 4, characterized in that The step of enriching the target glycoprotein / glycopeptide at the polypeptide level comprises: 3) The high mannose-type Afamin protein sample obtained in step 2 is enzymatically digested to obtain a peptide mixture; 4) The peptide mixture is enriched for N-linked glycopeptides of Afamin using an organic solvent method to obtain a serum protein sample that has been enriched.

6. The method according to claim 5, characterized in that The enzymatic cleavage method in step 3) is: adding the high mannose type Afamin protein sample to dithiothreitol for incubation, adding iodoacetamide for incubation, adding trypsin for incubation overnight, recovering the polypeptide, and vacuum freezing into dry powder to obtain a polypeptide mixture.

7. The method according to claim 5, characterized in that The enrichment method in step 4) is: resuspend the polypeptide sample, add 10 times the volume of 1% formic acid / acetone, vortex mix, incubate, and centrifuge to recover the precipitate; spin dry the supernatant using a vacuum centrifugal concentrator; use a mixture of acetonitrile, water and formic acid to dissolve the supernatant and precipitate to obtain a serum protein sample that has been enriched.

8. A method for detecting serum Afamin N-glycopeptide by LC-MS / MS, characterized in that: The method comprises the step of using the enriched serum protein sample obtained by the method according to any one of claims 1 to 7 to perform detection.

9. The method according to claim 5, characterized in that The method comprises the steps of performing LC-MS / MS analysis using an Easy-nLC 1200 liquid chromatograph and an Orbitrap Fusion mass spectrometer, The mobile phase A was 0.1% FA / H2O, the mobile phase B was 80% acetonitrile (ACN) / 0.1% FA, the pump flow rate was 2 μL / min, the sample was loaded onto the analytical column, and the separation flow rate was 300 nL / min; Liquid phase elution program: 0.0 ~ 3 min: 3% B ~ 8% B; 3 ~ 95 min: 8% B ~ 28% B; 95 ~ 110 min: 28% B ~ 32% B; 110 ~ 115 min: 32% B ~ 95% B; 115 ~ 120 min: 95% B; 120 ~ 120.1 min: 95% B ~ 3% B; The DDA-MS method was as follows: spray voltage: 1900 V, ion transfer tube temperature: 320 °C, mass spectrometry scan range (m / z) 375-1,500, MS1 resolution 60,000, maximum injection time 35 ms, AGC Target: 4e5; RF lens: 60%; precursor ion charge state: 2-7; dynamic exclusion cycle: 60 s; MS2 resolution 15000, ion fragmentation mode: HCD; maximum injection time 54 ms, AGC Target: 5e4.

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