A rapid method for analyzing proteins and highly polar long amino acid sequence glycopeptides

A rapid analysis, protein technology, applied in the field of glycopeptide analysis, can solve the problems of complete glycopeptide analysis efficiency and coverage reduction, sample loss, long time consumption, etc., and achieve the effect of high enrichment and identification efficiency

Active Publication Date: 2021-08-24
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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  • A rapid method for analyzing proteins and highly polar long amino acid sequence glycopeptides
  • A rapid method for analyzing proteins and highly polar long amino acid sequence glycopeptides
  • A rapid method for analyzing proteins and highly polar long amino acid sequence glycopeptides

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Embodiment 1

[0032] Sample protein preparation:

[0033] Preparation of cell / tissue protein: Use a cell protein reagent extraction kit (such as T-PER, RIPA) to extract cell protein, use as little lysate as possible to ensure a protein concentration of 2-10 mg / mL, and centrifuge at 14,000g for 5 minutes to remove cell debris . 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 separated protein (about 50μl), quantify it by Bradford method, and finally adjust the volume to 2mg / ml respectively.

[0035] ...

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 for 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 ...

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Abstract

The invention discloses a method for rapidly analyzing proteins and strong polar long amino acid sequence glycopeptides in biological samples, which includes the preparation of sample proteins; the preparation of a mixed chromatographic column for one-step enrichment method: mixing C18 and MAX fillers to prepare Hybrid Column; Protein Digestion; Glycopeptide Enrichment: Use the Hybrid Column to enrich glycopeptides; LC‑MS / MS analysis. The one-step enrichment method of the present invention has high enrichment and identification efficiency for long-sequence and more polar glycopeptides. Compared with the traditional sample preparation method, the one-step method of the present invention requires less sample preparation process and time. Requires less than 1 h of 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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Patent Type & Authority Patents(China)
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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