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Method and special apparatus for on-line enrichment and automatic analysis of endogenous polypeptide

An automatic analysis, endogenous technology, applied in the direction of measurement device, analysis material, material separation, etc., can solve the problems of difficult repetition in other laboratories, complicated system pipelines, large dead volume, etc., to achieve less sample consumption and simplify experiments. installation, eliminating time-consuming and labor-intensive effects

Inactive Publication Date: 2009-06-24
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Application Information

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Problems solved by technology

Since the polypeptide molecules are charged and can be retained on the ion-exchange medium, the ion-exchange restricted-access medium has attracted the attention of researchers in the enrichment of polypeptide samples. enrichment (Wagner, K.; Miliotis, T.; Marko-Varga, G.; Bischoff, R.; Unger, K.K. Anal. Chem. 2002, 74, 809-820), however the reported system is based on conventional liquid The phase chromato

Method used

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  • Method and special apparatus for on-line enrichment and automatic analysis of endogenous polypeptide
  • Method and special apparatus for on-line enrichment and automatic analysis of endogenous polypeptide
  • Method and special apparatus for on-line enrichment and automatic analysis of endogenous polypeptide

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Embodiment 1

[0032] 1. Preparation of serum samples: The serum samples were diluted 10 times with deionized water and then filtered with a 0.22 μm membrane for later use.

[0033] 2. Serum online enrichment and automatic analysis

[0034] (1) On-line enrichment and automatic analysis system: ①When the six-way valve is at the position of the dotted line, balance the automatic sampling system for 10 minutes under the condition that the flow rate on the analysis column is 200nL / min after splitting. At this time, the composition of the mobile phase was 0.1% formic acid; ②When the six-way valve was switched to the position of the solid line, 20 μL of the sample was uploaded to the limited-flow medium pre-column at a flow rate of 10 μL / min, and the injection time was 10 minutes. At this time, the proteins with large molecular weight are not retained, and all flow out from the emptying column, such as image 3 As shown in a; ③ When the six-way valve is switched to the dotted line position, the s...

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Abstract

The invention provides a method for on-line concentration and automatic analysis of endogenous polypeptides, and a special device. The method comprises the following steps: by utilizing the unique characteristic that a restricted access medium retains small molecular polypeptides and expels macromolecular proteins, the endogenous polypeptides are subjected to on-line concentration; a water solution, in which the volume concentration of formic acid or acetic acid is 0-5%, is used for leading a sample to pass through a pre-column of the restricted access medium for concentration; a buffer solution with the salt concentration being 10mM-2000mM and the pH value being 1-4 is used for eluting the sample concentrated in the pre-column to a reversed-phase column head; and the sample on the reversed-phase column head is subjected to separation and mass spectrometric detection with water/acetonitrile or water/methanol with the volume concentration ranging from 0% to 100%. By adopting the pre-column of the restricted access medium of a capillary and the stepwise gradient for elution, the invention achieves the effect of greatly simplifying the experimental facilities. The device can engage in on-line coupling with mass spectrum, and has the characteristics of simple operation, small sample consumption, the ability of carrying out multidimensional separation and the like.

Description

technical field [0001] The present invention relates to a method for on-line enrichment and automatic analysis of endogenous polypeptides in biological samples (body fluids, tissue extracts, etc.), and specifically develops a sample that is easy to operate, easy to use, and unsupervised The method of pretreatment and separation and detection integrates the methods of sample extraction and enrichment, chromatographic separation and mass spectrometry detection. Background technique [0002] Peptidomics is a research field developed in recent years (Schrader, M.; Schulz-Knappe, P. Trends Biotechnol. 2001, 19, S55-S60), because proteomics mainly focuses on proteins with a molecular weight of more than 10,000, while metabolic Omics focuses on small molecular metabolites with a molecular weight of less than 1,000. For a long time, peptides between the proteome and metabolome have been ignored by biologists as useless information. In 2002, the Lancet magazine published an article ...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/08
Inventor 邹汉法胡良海叶明亮蒋小岗
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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