Method for rapidly analyzing protein and strong-polarity long amino acid sequence glycopeptides in biological sample

A technology for rapid analysis of biological samples, applied in the field of glycopeptide analysis, can solve the problems of complete glycopeptide analysis efficiency and coverage reduction, time-consuming, sample loss, etc., and achieve high enrichment and identification efficiency

Active Publication Date: 2019-11-12
无锡麦迪科思生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The preparation process of complete glycopeptide samples of glycoproteins generally involves multiple steps. First, it undergoes enzymatic hydrolysis with proteolytic enzymes to cut the protein from a specific site into a polypeptide, and then desalt and dehydrate the polypeptide of the sample through a C18 hydrophobic column. After processing and freeze-drying, use lectin or hydrophilic enrichment method to enrich and separate glycopeptides. The whole process takes 3-5 days and takes a long time. Due to many steps, it will cause a large amount of sample loss. , leading to reduced analysis efficiency and coverage of intact glycopeptides

Method used

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  • Method for rapidly analyzing protein and strong-polarity long amino acid sequence glycopeptides in biological sample
  • Method for rapidly analyzing protein and strong-polarity long amino acid sequence glycopeptides in biological sample
  • Method for rapidly analyzing protein and strong-polarity long amino acid sequence glycopeptides in biological sample

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Experimental program
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Embodiment 1

[0032] Sample protein preparation:

[0033] Preparation of cell / tissue protein: use cell protein reagent extraction kit (such as T-PER, RIPA) to extract cell protein, try to use less lysate to ensure the protein concentration is 2-10mg / mL, and centrifuge at 14,000g for 5min to remove cell residues . The protein concentrations of the two cell extracts were determined with a BCA kit (guaranteed at least three repetitions).

[0034] Serum protein preparation: Serum samples were centrifuged at 12,000g for 15 minutes, and the middle liquid part was taken. Add to a 10KD ultrafiltration tube, place the ultrafiltration tube in a matching centrifuge tube, centrifuge at 12,000g for 15min, add 400μl of 40mM NH 4 HCO 3, centrifuge again, and repeat. Invert the filter membrane into a new centrifuge tube, centrifuge at 9000g for 3min, collect the isolated protein (about 50μl), quantify it with the Bradford method, and finally adjust the volume to 2mg / ml.

[0035] Preparation of exosome...

Embodiment 2

[0056] Analysis of WT and Fut8 knockout (Fut8 - / -) Complete glycopeptides of glycoengineered CHO cells:

[0057] We applied the one-step enrichment method of Example 1 to the analysis of wild-type (WT) and glycoengineered Chinese hamster ovary (CHO) cells, and evaluated the N-linked glycosylation and even Changes in glycoprotein expression. By knocking out the Fut8 gene in CHO cells (Fut8 - / - ) can generate core fucosylation-deficient cell lines. Fut8 (α1,6-fucosyltransferase) catalyzes the transfer of a fucose residue to position 6 of the innermost GlcNAc residue of an N-linked oligosaccharide of a glycoprotein (ie, core fucosylation). Based on the glycopeptides obtained by the one-step enrichment method, after LC-MS / MS analysis, the screening criteria of the database searched by GPQuest are as follows: (1) glycopeptide error rate (FDR) is less than 1%, (2) each peptide requires PSM ≥2, (3) all PSMs should annotate at least one N-linked glycan, (4) the core Fuc fragment i...

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Abstract

The invention discloses a method for rapidly analyzing protein and strong-polarity long amino acid sequence glycopeptides in a biological sample. The method comprises the following steps of preparingsample protein; preparing a mixed chromatographic column through a one-step enrichment method: mixing C18 and MAX filler to prepare the mixed chromatographic column; carrying out protein digesting; carrying out glycopeptides enriching: enriching glycopeptides by using the mixed chromatographic column; and carrying out LC-MS / MS analysis. According to the one-step enrichment method disclosed by theinvention, the enrichment and identification efficiencies of the glycopeptides with long sequence and stronger polarity are very high; and compared with a traditional sample preparation method, the method disclosed in the invention requires less sample preparation process and time, and only requires less than one hour of the preparation time before mass spectrometry analysis.

Description

technical field [0001] The invention belongs to the technical field of glycopeptide analysis methods, in particular to a method for rapidly analyzing proteins and strong polar long amino acid sequence glycopeptides in biological samples. Background technique [0002] As one of the most prevalent protein post-translational modifications, N-linked glycosylation plays a crucial role in many biological processes, especially protein folding in cells, cell adhesion, cell-matrix interaction, cell signaling , intracellular / extracellular targeting of organelles and pathogenesis of different diseases. Glycoproteins are widely distributed in organisms, such as tissue cells, various body fluids, etc. The complexity of protein glycosylation mainly includes the possible presence of multiple glycosylation sites in glycoproteins (macroscopic heterogeneity), the occupancy of glycosylation sites, and the differences in the glycan structure of each glycoside (microscopic heterogeneity) , pre...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06G01N30/08G01N30/28G01N30/86
CPCG01N30/02G01N30/06G01N30/08G01N30/28G01N30/8675
Inventor 杨刚龙陈文彦
Owner 无锡麦迪科思生物科技有限公司
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