Method for one-step desalting and enriching on low-abundance protein target

A low-abundance protein enrichment technology, applied in the field of biochemical analysis, can solve the problems of limited protein or peptide adsorption, insufficient salt elution, loss, etc., and achieve the effects of reduced sample area, improved sensitivity, and high sensitivity

Inactive Publication Date: 2006-08-02
FUDAN UNIV
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

The early practice was to use polymer coating as the adsorption stationary phase. Although the protein or peptide was adsorbed in a complex system and the interference of salt and surfactant was eliminated, the adsorption amount of protein or peptide was very limited, which did not meet the requirements of low abundance. Another traditional method is to use the imprinting method to self-assemble a layer of non-polar molecules on the surface of the non-spotted part of the metal target, and coat the polar polymer on the spotting part to spot the peptide sample. After covering the polar polymer coating, wash off the surfactant and salt with water, and finally add the matrix
In this way, not only the water washing process requires certain experience and technology, otherwise it will be insufficiently eluted or the sample will be eluted and lost, and it will affect the high throughput and high speed of the analysis process, which is not suitable for large-scale proteomics applications.

Method used

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  • Method for one-step desalting and enriching on low-abundance protein target
  • Method for one-step desalting and enriching on low-abundance protein target
  • Method for one-step desalting and enriching on low-abundance protein target

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Precisely position the perforated Kapton adhesive membrane by infrared laser to prepare the imprinted coating target plate of PMMA. The diameter of each imprinting spot is 990 microns, which is exactly half the diameter of the sample pool; 0.35 microliters of 60fmol / μL horse myocardium The erythrolytic peptide sample solution (solvent: 50% acetonitrile, 50% water, 0.1% trifluoroacetic acid) is dotted on the blotting coating and dried at room temperature; then the slightly excess volume is 0.7 microliters of organic matrix solution (α-CHCA, the solvent is 50% acetonitrile, 50% water, 0.1% trifluoroacetic acid) is applied to the surface of the imprinted coating, the excess solution will overflow the coating to the surrounding stainless steel area, and it will dry naturally at room temperature; the prepared sample It was sent to MALDI-TOF / TOF mass spectrometry (4700Proteomics Analyzer, Applied Biosystems) for detection. Mass spectrometry conditions: laser is Nd-YAG laser, wave...

Embodiment 2-4

[0033] Example 2-4 The influence of different polymer properties on MALDI-MS determination

[0034] Adjust the polymer coating substrate to PMMA-C 60 , PSt, PSt-C 60 , Other conditions remain unchanged, repeat the above desalination and enrichment experiments. See the attached Figure 8 Shown.

Embodiment 5-8

[0035] Examples 5-8 verify the influence of different polymer properties on MALDI-MS determination

[0036] Adjust the sample solution to 40fmol / μL bovine serum albumin hydrolyzed peptide solution (solvent is 50% acetonitrile, 50% water, 0.1% trifluoroacetic acid), and the polymer coating substrates are PMMA and PMMA-C respectively 60 , PSt and PSt-C 60 , Other conditions remain unchanged, repeat the above desalination and enrichment experiments. See the attached Figure 7 Shown.

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Abstract

The present invention relates to a method capable of implementing on-target one-step desalination and enrichment of trace protein or polypeptide by means of combined application of hydrophobic polymer microblotting technique and matrix auxiliary laser analysis ionization mass spectrography. Said microblotting hydrophobic polymer has good adsorption and enrichment effect for protein and polypeptide, so that it can implement one-step desalination and direct MALDI-TOF-MS analysis. Said invention can be extensively used in the field of protein phylogeny.

Description

Technical field [0001] The invention belongs to the technical field of biochemical analysis, and specifically relates to a method for desalination and enrichment of trace samples. Background technique [0002] With the smooth implementation of the Human Genome Project (HGP), people realized that functional genomics focuses on suggesting the biological functions of genes and clarifying the laws of gene activity at the overall level of the genome. Genes are only carriers of genetic information, and life activities The executor and embodiment of the gene is the expression product of the gene---protein. In the post-genomic era, the central task of life sciences is to clarify the expression rules and biological functions of all proteins that are expressed by the genome that actually perform life activities. That is, the research center of life sciences will shift from genomics to proteome. The proteome is much more complex than the genome. The development of proteomics requires not on...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/08G01N30/72
Inventor 贾韦韬陆豪杰吴慧霞蔡瑞芳杨芃原
Owner FUDAN UNIV
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