Method for fast enriching trace polypeptide and protein and realizing identification

A protein and enrichment technology, applied in the field of biochemical analysis, can solve the problems of easily polluting the ion optical channel of mass spectrometry, affecting signal stability and reproducibility, and long enrichment time, so as to ensure reproducibility and stability, avoid Sample loss, good mass spectrum reproducibility effect

Inactive Publication Date: 2006-08-02
FUDAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the enrichment time of the above method is long, up to 90 minutes; and the solid particles brought into the mass spectrometer will affect the signal stability and reproducibility on the one hand, and on the other hand, the ion optical channel of the mass spectrometer is easily polluted by the sputtering of nanoparticles.

Method used

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  • Method for fast enriching trace polypeptide and protein and realizing identification
  • Method for fast enriching trace polypeptide and protein and realizing identification
  • Method for fast enriching trace polypeptide and protein and realizing identification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] CaCO 3 -Study on the adsorption behavior of PMMA materials to peptides

[0027] Prepare 1 fmol / μL level of peptide mixture (standard peptides 1, 2, 3 dissolved in 50% ACN50% H 2O), take 0.5 μL solution and mix it with an equal volume of α-CHCA matrix solution (50% acetonitrile aqueous solution of 0.1% TFA) on the MALDI target plate, carry out MALDI-TOF / MS analysis after drying and crystallization, see figure 2 a. nanoCaCO 3 -PMMA material was incubated in the above solution for 10 minutes, centrifuged for 10 minutes, discarded the supernatant, then took 5 μL of acetonitrile solution containing acetic acid (0.2 μL) to re-mix the stationary phase, centrifuged for 5 minutes, and took the supernatant (final ACN supernatant solution containing peptide / PMMA chain) 0.5 μL and equal volume of α-CHCA matrix solution (50% acetonitrile aqueous solution of 0.1% TFA) were mixed and dried on the MALDI target plate and then MALDI-TOF / MS analysis was carried out. See figure 2 b....

Embodiment 2

[0028] Example 2CaCO 3 -Study on the adsorption of cytochrome c protein by PMMA material

[0029] Adjust the sample solution to 500 fmol / μL cytochrome c protein solution (50% ACN50% H 2 O), other conditions remain unchanged, repeat above-mentioned enrichment experiment. The results obtained can be found in the appendix image 3 shown.

Embodiment 3

[0030] Example 3CaCO 3 -Study on the adsorption of PMMA material to horse myoglobin

[0031] Adjust the sample solution to 600fmol / μL horse myoglobin solution (50%ACN50%H 2 O), other conditions remain unchanged, repeat above-mentioned enrichment experiment. The results obtained can be found in the appendix Figure 4 shown in a and c. Figure 4 b is nano CaCO 3 - PMMA material was incubated in the above solution for 10 minutes and then centrifuged for 10 minutes, then 0.5 μL of the supernatant was mixed with an equal volume of α-CHCA matrix solution (0.1% TFA in 50% acetonitrile aqueous solution) on a MALDI target plate and dried after crystallization Results of MALDI-TOF / MS analysis.

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Abstract

The present invention relates to a method for quickly enriching trace polypeptide or protein sample by using nano calcium carbonate particles in-situ polymerized polymethyl methacrylate (CaCo3-PMMA) as nano adsorbing agent and utilizing solid-free miniaturated matrix auxiliary laser analysis ion source mass spectrography to make direct analysis. The invented nano material not only can implement quick and high-effective enrichment of protein and polypeptide, but also has good compatibility with matrix auxiliary laser analysis ion source mass spectrography. Said invention can be extensively used in the field related to protein phylogeny.

Description

technical field [0001] The invention belongs to the technical field of biochemical analysis, in particular to a method for rapidly enriching and identifying trace samples of polypeptides and proteins. Background technique [0002] In the post-genome era, proteome research has attracted more and more attention from scientists at home and abroad, and the invention and successful application of matrix-assisted laser desorption ion source (MALDI) has made biological mass spectrometry an important technology in proteomics research. platform. The analysis and identification of low-abundance proteins is one of the key and difficult aspects of proteomics research. Proteins that undertake important life activities in organisms are often low-abundance proteins, but their extremely low content brings difficulties to subsequent analysis and detection, and limits people's research and understanding of them. Therefore, the effective concentration of low-abundance proteins is one of the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/08G01N30/72
Inventor 贾韦韬陆豪杰陈雪花杨芃原
Owner FUDAN UNIV
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