Quantitative analysis method for intact protein under different physiological or pathological conditions

A quantitative analysis and protein technology, applied in the field of proteomics and systems biology, can solve the problems of limited application range, and achieve the effect of high labeling efficiency and high accuracy

Inactive Publication Date: 2016-01-13
TONGJI UNIV
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Problems solved by technology

In terms of overall protein quantitative labeling, SILAC-based in vivo labeling and TMT-based in vitro labeling, due to incomplete labeling of targeted amino acids (such as incomplete s

Method used

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  • Quantitative analysis method for intact protein under different physiological or pathological conditions
  • Quantitative analysis method for intact protein under different physiological or pathological conditions

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Embodiment

[0021] A method for the quantitative analysis of whole proteins under different physiological or pathological conditions, using such as figure 1 The overall process shown includes the following steps:

[0022] (1) The overall protein of two different physiological or pathological conditions (take the control group and the disease group as an example) is first reduced with dithiothreitol and alkylated with iodoacetamide;

[0023] (2) The above two groups of alkylated proteins were uniformly guanidinated with O-methylisourea (2M, 100mM sodium bicarbonate solution) on the ε-amino group on the lysine residue in the protein sequence (use 2M NaOH to adjust the pH to 11, 65°C), quenched with 10% (v / v) trifluoroacetic acid solution after 15 minutes of reaction;

[0024] (3) The two groups of proteins after protection were respectively treated with formaldehyde and sodium deuterated cyanoborohydride, 13 C-labeled formaldehyde and sodium cyanoborohydride are used to label the N-termin...

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Abstract

The invention relates to a quantitative analysis method for intact protein under different physiological or pathological conditions. The method comprises firstly taking two groups of intact protein under different physiological or pathological conditions respectively as a control group and a disease group, and performing reduction and alkylation; performing chemical protection on epsilon-amino on lysine residue in the two groups of the alkylated protein sequences; performing isobaric marking on the N-end amino of the two groups of protein subjected to chemical protection; mixing the two groups of protein subjected to isobaric marking according to equal proportions, and performing high-resolution mass spectrometry and tandem mass spectrometry analysis to obtain a data set; and performing qualitative and quantitative database search on the data set, so as to obtain protein ID and the relative proportion of each protein relative to the protein in the control group, in other words, the up-regulation or down-regulation situations of all protein under disease conditions. Compared with the prior art, the method is high in marking efficiency and high in accuracy, and is suitable for quantitative analysis on intact protein based on high-resolution tandem mass spectrometry.

Description

technical field [0001] The present invention relates to a method for analyzing protein molecules, in particular to a method for quantitatively analyzing whole proteins under different physiological or pathological conditions, and relates to the technical fields of systems biology and proteomics related to biological mass spectrometry. Background technique [0002] In the past two or three years, commercial mass spectrometers with high-quality resolution and high-quality measurement accuracy have developed by leaps and bounds, that is, the emergence of orbitrap mass spectrometers. Orbitrap mass spectrometers have the same resolution and accuracy as Fourier transform ion cyclotron resonance mass spectrometers; but they are relatively cheap, with faster mass spectrometry acquisition and higher dissociation efficiency. The relative popularity of this mass spectrometer provides a solid foundation for the mass spectrometry and tandem mass spectrometry analysis of the overall prote...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/6848
Inventor 田志新肖开捷方后琴沈赟王悦
Owner TONGJI UNIV
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