Method and reagent kit for enriching and sequencing peptide fragment containing histidine

A technology of histidine peptide and histidine, which is applied in the direction of measuring devices, material separation, instruments, etc., can solve the problems of application limitations, complex cracking behavior of mass spectrometry, difficulty in automatic online coupling, etc., and achieve the effect of wide practicability

Inactive Publication Date: 2007-09-26
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

Due to the defects in the separation technology itself or the connection between separation and subsequent identification, such as the resolution and recovery rate of protein chromatographic separation are not high, two-dimensional electrophoresis is not suitable for separating proteins with strong hydrophobicity or extreme isoelectric point and molecular weight, and it is not easy to To realize the automation of operation and online coupling with mass spectrometry, the application of the "top-down" strategy is subject to many restrictions
More importantly, the mass spectrometry fragmentation behavior of these unmodified peptides is relatively complex, which affects the quality of their secondary mass spectra and the analysis of their sequences

Method used

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  • Method and reagent kit for enriching and sequencing peptide fragment containing histidine
  • Method and reagent kit for enriching and sequencing peptide fragment containing histidine
  • Method and reagent kit for enriching and sequencing peptide fragment containing histidine

Examples

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Effect test

Embodiment 1

[0031] Example 1 Enrichment and sequencing of histidine-containing peptides in horse myoglobin

[0032] 1.1 Take 1mg of horse myoglobin and dissolve it in 1mL of 0.2M pH 8 borate solution. Myoglobin does not contain cysteine ​​residues, so the reductive alkylation step can be omitted. Add 20ug trypsin, digest at 37°C for 12 hours. Add 0.1M o-methylisourea, adjust the pH to 11 with sodium hydroxide, react at 37°C for 4 hours, then adjust the pH to 8 with hydrochloric acid, add 20mM o-sulfobenzoic anhydride, and react at room temperature for 1 hour.

[0033] 1.2 On the Elite P230 liquid chromatograph (Dalian Elite Company), use a C18-reversed-phase chromatographic column (200×4.6mm, 5um, 300 Ȧ, Dalian Elite Company) and a strong cation exchange column (200×4.6 mm, 5um, 300 Ȧ, Dalian Elite Company) in series for the enrichment of histidine peptides. First equilibrate the chromatographic system with 0.1% trifluoroacetic acid (TFA), wash the column with 10 times the column volum...

Embodiment 2

[0035] Example 2 Enrichment and sequencing of histidine-containing peptides in bovine lactoglobulin

[0036] 2.1 Take 1mg of bovine lactoglobulin and dissolve it in 0.1mL of 0.2M borate solution of pH 8 (containing 6M guanidine hydrochloride). Add 10mM dithiothreitol (DTT), react at 37°C for 1 hour, cool to room temperature, add 20mM iodoacetamide (IAA), react at room temperature for 1 hour, and then add 10mM DTT to terminate the reaction. After diluting 6 times with water, add 20ug trypsin, and digest at 37°C for 12 hours. Add 0.1M o-methylisourea, adjust the pH to 11 with sodium hydroxide, react at 37°C for 4 hours, then adjust the pH to 8 with hydrochloric acid, add 20mM o-sulfobenzoic anhydride, and react at room temperature for 1 hour.

[0037] 2.2 On the Elite P230 liquid chromatograph (Dalian Elite Company), use a C18-reversed-phase chromatographic column (200×4.6mm, 5um, 300 Ȧ, Dalian Elite Company) and a strong cation exchange column (200×4.6 mm, 5um, 300 Ȧ, Dalian ...

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Abstract

This invention provides one new method and agent case for collecting and testing group aminoacidemia peptides, which comprises the following steps: protein restores alkyl base and tryptone protein enzyme cut, peptides section N end amidogen decoration and group aminoacidemia peptides color spectrum collection and mass spectrum testing sequence; identifying the protein with peptides sections in mixture objects.

Description

technical field [0001] The invention relates to a protein sequencing method and kit; specifically, the invention relates to a method and kit for chromatographic enrichment and mass spectrometry sequencing of histidine-containing peptides. Background technique [0002] With the development of mass spectrometry technology, biological mass spectrometry plays an increasingly important role in the sequencing and identification of proteins. However, due to the large size of protein molecules, it is difficult for existing mass spectrometers to identify unknown proteins by directly sequencing the entire protein molecule. Instead, the protein molecule is first cut into smaller peptides by chemical or enzymatic methods, and then the peptides are sequenced. Perform sequencing. And because the proteome contains many kinds of proteins, the system is very complex, and must be separated before mass spectrometry analysis to reduce the complexity of the system. According to the sequence of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06C12Q1/68B01D15/08
Inventor 钱小红周春喜张养军
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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