Polypeptide mixture isoelectric focusing separation method for proteomics analysis

Abstract

The invention discloses a polypeptide mixture isoelectric focusing separation method for proteomics analysis. The method comprises the following steps: performing restriction enzyme digestion on a total protein mixture of a biological sample completely to obtain relatively short peptide fragment mixtures; re-dissolving the peptide fragment mixtures by using a peptide fragment isoelectric focusing buffer solution; performing isoelectric focusing electrophoresis in a loading manner by adopting a loading cup, so as to realize the effective separation of a complex peptide fragment mixture; performing desalination on a taken polypeptide mixture pre-separated from each groove and performing mass spectrometry analysis so as to obtain more polypeptide signals, higher peptide fragment covering rate and higher protein identification number compared with those obtained when a conventional method is adopted. The method is precise, efficient and low in cost, can be used in multidimensional liquid chromatography-mass spectrum identification technology systems of total proteins of the various biological samples, can replace a conventional ion exchange chromatography pre-separation method so as to realize the efficient pre-separation of the peptide fragment mixtures, and avoids the lost of peptide fragments, and therefore, proteome expression profile information with the high covering rate can be established.

Description

technical field [0001] The invention belongs to the technical field of proteomics, and in particular relates to an isoelectric focusing separation method for a polypeptide mixture used for proteomics analysis. Background technique [0002] Proteins are the key structural and functional molecules in organisms, the executors of life activities and the embodiment of life phenomena. Proteome refers to all proteins expressed by genes in a cell or a tissue under specific conditions. The analysis and identification of these proteins is called proteomics. [0003] At present, there are two main types of proteomics research methods, namely two-dimensional electrophoresis (Two-dimensional polyacrylamide gel electrophoresis, 2DE) technology and multi-dimensional liquid chromatography (Multi-dimensional liquid chromatography, MDLC) technology. Two-dimensional electrophoresis-mass spectrometry is a classical protein research method widely used at present. First, the complex protein mixt...

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Problems solved by technology

[0005] (2) The operation of the experiment is cumbersome, and the reproducibility of the experimental results is poor;

[0006] (3) For strongly acidic proteins, strongly basic proteins, hydrophobic proteins and low-abundance proteins, 2D-PAGE cannot detect them well

[0007] (4) 2D-PAGE cannot be connected online with mass spectrometry to achieve efficient protein separation and identification

However, when the peptide mixture is separated in the first dimension, if these modes are used, some peptides will be lost with the elution of the mobile phase, which will affect the detection of low-abundance proteins.

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