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Dual quantification method and reagent kit for stable isotope 18 O marked proteome

A stable isotope and proteome technology, applied in measuring devices, biological testing, material inspection products, etc., can solve the problem of high price of Lys-N enzyme, and achieve the effect of wide practicability, easy realization and simple operation

Inactive Publication Date: 2008-05-07
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

The disadvantage of this method is that the Lys-N enzyme is more expensive

Method used

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  • Dual quantification method and reagent kit for stable isotope 18 O marked proteome
  • Dual quantification method and reagent kit for stable isotope 18 O marked proteome
  • Dual quantification method and reagent kit for stable isotope 18 O marked proteome

Examples

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Embodiment 1

[0036] Example 1 Double quantitative labeling of horse myoglobin

[0037] 1.1 Take 0.1 mg of horse myoglobin and dissolve it in 100 μL of 50 mM pH7.0 triethylamine bicarbonate buffer solution. Denature the protein by heating at 95°C for 5 minutes. Myoglobin has no cysteine ​​residues in its amino acid sequence, so a reductive alkylation step is not required. Add 2ug trypsin, digest at 37°C for 2 hours. Then add 2ug of trypsin, and digest at 37°C for 10 hours. Divide the myoglobin peptides obtained by enzymatic digestion into four parts, two of which were heated in a boiling water bath for ten minutes and then cooled rapidly in an ice bath to inactivate trypsin. Then dry completely in a vacuum dryer with the remaining two copies. In the obtained solid powder, take one part of trypsin-inactivated and one part of trypsin-inactivated sample and dissolve it in 25 μL 18 50mM triethylamine bicarbonate buffer solution with pH 7.0 in O water. The remaining two aliquots were disso...

Embodiment 2 6

[0042] The double quantification of embodiment 2 six kinds of standard protein mixtures

[0043] 2.1 Six standard protein mixtures were purchased from Sigma, USA, and configured according to the following proportions (mixtures A and B, μg / mL): β-casein (10, 10); β-lactoglobulin (10, 40) ; myoglobin (60, 20); bovine serum albumin (40, 20); lysozyme (50, 50); transferrin (30, 60). The protein mixture was dissolved in 50 μL pH7.0, 50 mM triethylamine bicarbonate solution, and denatured by heating at 95 ° C for 5 min. Add 2 μL of 50 mM reducing agent tricarboxyethyl phosphine, and incubate at 60° C. for 1 h. Then 2 μL of 250 mM alkylating reagent iodoacetamide was added, left in the dark for 10 minutes, and trypsin was added according to the ratio (1:20w / w), 37°C, 18h. Vacuum-dry the protein peptide solution obtained by enzymatic digestion, add 18 The triethylamine bicarbonate buffer solution prepared with O water was dissolved, and reacted with 5-fold excess bis-acid anhydride...

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Abstract

The invention provides a new method for double quantifying the protein group marked on the basis of stable oxygen isotope <18>O, which is characterized in that: based on the fact that the N end peptide section and the C end peptide section are generally labeled, and by means of combining the <18>O chemical labeling and enzyme digestion, the quantifying strategy for two <18>O markings of the same sample can be accomplished. The method relates to the reductive alkylation and enzyme digestion of protein, dual-functional reagent modifying and <18>O marking of peptides, multi-dimensional liquid phase chromatogram separation and multistage Tandem Mass Spectrometry quantification and identification. A first mass spectrum peak area is used to quantify; and the identification of the protein is conducted through a second stage mass spectrum and by searching the database. The invention also provides a reagent box for facilitating the implementation of the method.

Description

technical field [0001] The invention relates to a method and kit for double quantification of proteins in proteomics. Includes a stable isotope for quantitative proteomics 18 Novel method and kit for O-labeling proteins chemically labeled with dianhydride reagents. Background technique [0002] Proteomics has become an important content of current life science research. Quantitative or differential proteome research to study the composition and changes of cellular proteins under different physiological and pathological conditions on a large scale has become a hot and difficult point in proteomics research. Accurate quantification in proteome research has posed new challenges to current proteome quantification methods and techniques, and has attracted more and more attention from researchers from all over the world. [0003] At present, large-scale proteome quantification methods mainly rely on 2DE staining methods and stable isotope-labeled mass spectrometry detection tec...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N30/00
Inventor 钱小红刘慧玲张养军
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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