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A Quantitative Analysis Method Integrating Proteome and Glycoproteome

A proteome and quantitative analysis technology, applied in the field of biochemistry, can solve the problems of lack of integrated proteome and glycoproteome quantitative analysis methods, loss of specific information of glycosylation sites, and low abundance of glycosylated peptides. , to achieve the effect of simple and time-saving steps, reliable quantitative results, and avoiding the influence of sialic acid

Active Publication Date: 2022-06-21
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, there is currently a lack of quantitative analysis methods that integrate proteome and glycoproteome, because there are many difficulties and challenges in technical methods
[0004] First of all, in the sample pretreatment of the proteome alone, due to the low abundance of glycosylation modifications, the signal of glycopeptides in mass spectrometry detection will be suppressed by non-glycopeptides, so only qualitative and quantitative analysis of proteins can be performed
[0005] Secondly, in the sample pretreatment of the glycoproteome alone, the abundance of glycosylated peptides is very low, so an effective glycopeptide enrichment method is required to enrich glycopeptides and remove the interference of non-glycopeptides, so in mass spectrometry detection Only information about glycopeptides can be obtained
[0006] Finally, traditional collision-based mass spectrometry fragmentation methods such as collision fragmentation (CID) will preferentially fragment sugar chains, which can only produce few peptide backbone fragments and low glycoxonium ions, which are critical for the identification of intact glycopeptides. is very difficult, so it is necessary to find alternative fragmentation methods, such as step energy HCD fragmentation mass spectrometry (SCE-HCD-MS / MS)
[0007] In addition, the research on the proteome and the glycoproteome in the prior art is mainly done separately, and there are few reports on the integrated quantitative analysis of the proteome and the glycoproteome (complete N-glycopeptide and desialic N-glycopeptide)
Zhang Hui et al. established an analysis method integrating proteome and glycoproteome to study prostate cancer cell lines, but the proteome quantification method is quantified by iTRAQ markers, and the glycoproteome is deglycosylated glycopeptide, so the cost of this method is high , long time, and lost glycosylation site-specific information

Method used

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  • A Quantitative Analysis Method Integrating Proteome and Glycoproteome

Examples

Experimental program
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Effect test

Embodiment 1

[0058] Example 1 Preparation of proteomic and glycoproteomic samples of protein IgG

[0059] 1. Method

[0060] (1) Dissolve 100 μg of commercial human IgG (Sigma) in 200 μL of 50 mmol / L ABC solution, and heat at 95° C. for 10 min for denaturation.

[0061] (2) DTT with a final concentration of 20 mmol / L was added to react at 56°C for 45 min, and IAM with a final concentration of 50 mmol / L was added to react for 1 h at room temperature in the dark.

[0062] (3) All the reaction solution was added into a 10KD ultrafiltration tube, centrifuged at 13000g for 15min×3 times, and washed with 200μL of 50mmol / L ABC each time.

[0063] (4) Add 100 μL of 50 mmol / L ABC and 2 g of trypsin (Promega), continue the reaction in a shaker at 37 °C for 2 h, centrifuge at 13,000 g for 15 min × 3 times, collect the peptide fragments, take 1 μg for storage, and the rest after lyophilization- Reserve at 80°C.

[0064] (5) Weigh 5 mg of Venusil HILIC filler (Bonna Aijer), rinse the filler with 0.1...

Embodiment 2

[0074] Example 2 Proteome and Glycoproteome Mass Spectrometry Analysis and Data Processing of Protein IgG

[0075] 1. Method

[0076] (1) Take 2 μL of the peptides, intact N-glycopeptides, and asialo-N-glycopeptides from steps (4) (8) (9) in Example 1 for mass spectrometry analysis. The instrument was an Orbitrap Fusion Lumos mass spectrometer.

[0077] (2) The chromatographic conditions are: chromatographic column: The inner diameter of the Magic C18 column is 75 μm, the length is 15 cm, and the particle size of C18 is 3 μm. Mobile phase A solution: 0.1% (v) trifluoroacetic acid in water; Mobile phase B solution: 0.1% (v) trifluoroacetic acid in 80% (v) acetonitrile solution; Mobile phase B linear gradient: 5~32% ; Flow rate of mobile phase: 0.3 μL / min.

[0078] (3) The mass spectrometry conditions for proteome analysis were: the fragmentation mode of the mass spectrometer was HCD, the time was 78 min, and the flow rate was 0.3 μL / min. The mass range of the primary mass s...

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Abstract

The invention provides a quantitative analysis method for integrating proteome and glycoproteome, which comprises the following steps: (1) performing reductive alkylation treatment after heating and denaturing the protein; (2) using trypsin in an ultrafiltration tube to convert the protein Cut peptides with enzymes and centrifuge to obtain peptides; (3) Hydrophilic interaction chromatography HILIC filler binds peptides, washes and removes impurities, and then elutes to obtain complete N-glycopeptides; (4) Adds trifluoroacetic acid and heats to remove N-glycopeptides ‑Sialic acid on the periphery of the sugar chain to obtain asialic N‑glycopeptide; (5) Use HCD‑MS / MS to analyze the peptide to obtain proteome data, and use MaxQuant for qualitative and quantitative analysis; (6) Use SCE‑HCD‑MS / MS analysis of intact N-glycopeptides and asialo-N-glycopeptides, using Xcalibur software for qualitative and quantitative analysis. The invention can simply, quickly and effectively realize the quantitative analysis of proteome and glycoproteome, and has a good application prospect in the study of the occurrence and development mechanism of diseases and the discovery of new biomarkers.

Description

technical field [0001] The invention relates to the field of biochemistry, in particular to a quantitative analysis method integrating proteome and glycoproteome. Background technique [0002] It has been found that important proteins and their glycosylation modifications in a large number of disease patients will undergo abnormal changes, such changes include protein type, protein expression, sugar chain structure, glycosylation modification sites, and degree of glycosylation modification. Therefore, in-depth understanding of these important proteins and their glycosylation changes, the development of new methods for simultaneous analysis of proteome and glycoproteome, and the acquisition of multi-dimensional information will help to understand the mechanism of disease occurrence and development and provide diagnostic markers and therapeutic targets. [0003] However, there is currently a lack of quantitative analysis methods that integrate proteome and glycoproteome due to...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68
CPCG01N33/6845
Inventor 张勇赵婉君杨浩程惊秋
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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