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Whole protein online fractionation pre-separation and separation analysis method and its liquid chromatography system

A separation analysis and pre-separation technology, which is applied in the direction of material separation, analysis materials, measuring devices, etc., can solve the problems of slow analysis speed, low resolution, low sensitivity, etc., and achieve high speed, high system flexibility, and separation resolution maximized effect

Inactive Publication Date: 2014-10-22
TONGJI UNIV
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  • Application Information

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Problems solved by technology

Since the sample loss in the desalting process is very serious and time-consuming, the analysis speed is slow and the sensitivity is low when ion exchange chromatography is used for pre-separation
The eluent of size exclusion chromatography is compatible with liquid chromatography-mass spectrometry analysis, can be used online, and the analysis speed is fast, but its resolution is often very low

Method used

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  • Whole protein online fractionation pre-separation and separation analysis method and its liquid chromatography system
  • Whole protein online fractionation pre-separation and separation analysis method and its liquid chromatography system
  • Whole protein online fractionation pre-separation and separation analysis method and its liquid chromatography system

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Embodiment

[0053] The working process of the liquid chromatography system will be described below by taking the separation and analysis of the whole proteome of Escherichia coli as an example.

[0054] Step 1, sample injection: in the injection valve, the protein sample is injected into the sample loop by the syringe, such as figure 1 shown;

[0055] Step 2, pre-separation: Mobile phase A (100% water + 0.1% formic acid) carries the sample in the sample loop through T1, T2, T3, T4 and T5 trapping columns in sequence, such as figure 2 shown;

[0056] Step 3, C1 separation: The sample trapped on the T1 trapping column (filled with C1 stationary phase) is separated by the A1 analytical column (filled with C1 stationary phase) and detected by mass spectrometry. The connection positions of each chromatographic valve are as follows: image 3 Shown; The chromatogram obtained by analyzing the protein trapped on the T1 trapping column (packed with C1 stationary phase) by A1 analytical column (p...

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Abstract

The invention provides an on-line pre-separating and separating analysis method for a whole protein and a liquid chromatogram system thereof. By utilizing orderly proportionately increased characteristic of hydrophobicity of Cn capture column, proteins in a protein mixture are orderly gradingly captured from large to small to implement pre-separation; and the proteins on the capture column are orderly matched by relative Cn analytical columns to implement secondary separation, and the analytical column is used together with the mass spectrum for analysis. The orderly proportionately increased capture columns Cn (n=1-18 or partial combination thereof, such as 1, 2, 4, 8 and 18) are used for on-line pre-separating samples, which greatly raises a pre-separation speed, sensitivity and resolution; and the separation columns and the capture column are pairwise matched (such as C4 capture and C4 separation column are matched), which have consistent hydrophobicity, thereby implementing maximization of separation resolution.

Description

technical field [0001] The invention relates to a technology and method for on-line fractionation, pre-separation and separation analysis of whole proteins. Background technique [0002] In today's post-genome era, the entire genome sequences of many species have been or are being determined, which makes comprehensive and in-depth research on the proteome possible. Since most of the biological functions are ultimately transmitted and realized through proteins, the research on proteins will promote the development of biology and medicine more directly and rapidly on the basis of gene research. The technology based on high performance liquid chromatography-mass spectrometry can quickly and sensitively analyze and identify proteins and their mixtures, and is the frontier and preferred research method for protein research. In the past ten years, mass spectrometry-based protein analysis and identification technology has been developed rapidly, and it has been able to perform com...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02G01N30/06
Inventor 田志新
Owner TONGJI UNIV
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