Method for qualitative and quantitative analysis of non-enzymatic glycosylated plasma albumin

A technology of plasma albumin and non-enzymatic glycosylation, which is applied in the field of qualitative and quantitative analysis of non-enzymatic glycosylated plasma albumin, can solve the problem of inability to provide information on the absolute content of glycosylation sites, complex glycosylation states, different And other issues

Inactive Publication Date: 2019-07-16
SHANDONG UNIV
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Problems solved by technology

However, because there are a large number of potential glycosylation sites on HSA and the glycosylation state is complex, the glycosylation sites of diabetic patients are not completely consistent with the glycosylation sites of normal people, and the degree of glycosylation at the same site is also different. are very different, so HSA total glycosylation levels do not provide sufficient information to assess the condition of diabetic patients
[0004] Literature Andrej Frol

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  • Method for qualitative and quantitative analysis of non-enzymatic glycosylated plasma albumin
  • Method for qualitative and quantitative analysis of non-enzymatic glycosylated plasma albumin
  • Method for qualitative and quantitative analysis of non-enzymatic glycosylated plasma albumin

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Embodiment Construction

[0019] A method for qualitative and quantitative analysis of non-enzyme glycosylated plasma albumin, comprising the following steps:

[0020] (1) Add PEG-6000 to the plasma, use PEG precipitation to remove the globulin in the plasma, and keep the supernatant containing plasma albumin for later use; qualitative analysis enters step (2), and quantitative analysis enters step (3);

[0021] (2) Separation of glycosylated HSA by affinity chromatography;

[0022] (3) Reductive alkylation of HSA;

[0023] (4) Add trypsin to the protein solution after reductive alkylation, the mass ratio of trypsin to protein solution is 1:25, and incubate overnight at 37° C.; qualitative analysis enters step (8), and quantitative analysis enters step ( 5);

[0024] (5) labeling the enzymatic HSA used for quantitative experiments;

[0025] (6) using an affinity chromatography column to separate the labeled glycosylated HSA peptides;

[0026] (7) C18Ziptip desalting; Quantitative analysis enters st...

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Abstract

The invention relates to a method for qualitative and quantitative analysis of non-enzymatic glycosylated plasma albumin. After reductive alkylation of plasma albumin and trypsin enzymolysis, non-enzymatic glycosylation of the plasma albumin is analyzed qualitatively and quantitatively by nanoliter liquid chromatography-multistage mass spectrometry (nL-MSn) and isobaric tags for relative and absolute quantitation (iTRAQ). The plasma albumin is degraded into peptide fragments by reductive alkylation and trypsin enzymolysis, amino acid sequence and glycosylation site information are obtained byhigh-resolution mass spectrometry after nanoliter liquid phase separation, and accurate quantification of the glycosylation site is achieved by iTRAQ reporter ions. The invention realizes the comprehensive qualitative and quantitative analysis of the glycosylation site of plasma albumin and can be used for the monitoring of blood glucose concentration and the detection of early diabetes.

Description

technical field [0001] The invention relates to a method for qualitative and quantitative analysis of non-enzymatic glycosylated plasma albumin, in particular to a method for reducing plasma albumin to alkylation and enzymolysis with trypsin, and then performing "nanoliter liquid chromatography-multistage mass spectrometry ( nL-MSn) combined with isotope-labeled relative and absolute quantification (iTRAQ)" is a method for qualitative and quantitative analysis of non-enzymatic glycosylation of plasma albumin, which belongs to the field of pharmaceutical analysis technology. Background technique [0002] Diabetes mellitus is a common chronic disease in which chronic hyperglycemia leads to various pathological changes, including excessive production of advanced glycation end products (AGEs). AGEs is a general term for proteins, lipids, and nucleic acids that undergo irreversible covalent modification by reducing sugars or sugar derivatives without the participation of glycosyl...

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Application Information

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IPC IPC(8): G01N30/02G01N30/06G01N30/72
CPCG01N30/02G01N30/06G01N30/72G01N2030/067
Inventor 迟连利崔雪莹仇红燕张群业王哲张馨洁
Owner SHANDONG UNIV
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