Alkylation derivatization reagent and application of alkylation derivatization reagent in peptide fragment marking and mass spectrometric detection

A derivative reagent and mass spectrometry detection technology, which is applied in the field of biomacromolecule labeling, can solve the problems of limited improvement in the detection sensitivity of peptide mass spectrometry, and achieve the effects of lower cost, increased charge number, and improved detection rate

Inactive Publication Date: 2015-04-08
HEBEI UNIVERSITY
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  • Application Information

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Problems solved by technology

[0005] Although a variety of alkylation derivatization reagents have been reported and applied to the alkylation derivatization of cysteine ​​peptides, however, the sensitivity improvement of mass spectrometry detection of peptides is relatively limited, and it is currently only used in synthetic peptides or standard proteins Analysis of enzymatic hydrolysis products

Method used

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  • Alkylation derivatization reagent and application of alkylation derivatization reagent in peptide fragment marking and mass spectrometric detection
  • Alkylation derivatization reagent and application of alkylation derivatization reagent in peptide fragment marking and mass spectrometric detection
  • Alkylation derivatization reagent and application of alkylation derivatization reagent in peptide fragment marking and mass spectrometric detection

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Effect test

Embodiment 1

[0043] Tris-HCl buffer solution with a concentration of 50mmol / L and a pH of 8.4 was added to the peptide ALVCEQEAR to prepare a peptide solution with a concentration of 0.33mg / mL. Take 15 μL of the peptide solution, add 10 μL of tris(2-carboxyethyl)phosphine hydrochloride solution with a concentration of 20 mmol / L, and react at 50° C. for 1 hour. After cooling to room temperature, 50 μL of IPBI with a concentration of 50 mmol / L was added to the above solution, and the reaction was continued at 37° C. for 2 hours. After the reaction was completed, samples were directly sampled for MALDI-TOF MS analysis. The results showed that the derivation efficiency of the peptide ALVCEQEAR was 100% ( figure 1 Middle A panel).

Embodiment 2

[0045] Add 50 mmol / L Tris-HCl buffer solution with a pH of 8.4 to the peptide CDPGYIGSR to prepare a 0.33 mg / mL peptide solution. Take 15 μL of the peptide solution, add 10 μL of tris(2-carboxyethyl)phosphine hydrochloride with a concentration of 20 mmol / L, and react at 50° C. for 1 hour. After cooling to room temperature, 50 μL of IPBI with a concentration of 50 mmol / L was added to the above solution, and the reaction was continued at 37° C. for 2 hours. After the reaction was completed, samples were directly sampled for MALDI-TOF MS analysis. The results showed that the derivatization efficiency of the peptide CDPGYIGSR was 99.4% ( figure 1 Middle B panel).

Embodiment 3

[0047] Tris-HCl buffer solution with a concentration of 50mmol / L and a pH of 8.4 was added to the peptide CKDECSLDG to prepare a 0.33mg / mL peptide solution. Take 15 μL of the peptide solution, add 10 μL of tris(2-carboxyethyl)phosphine hydrochloride solution with a concentration of 20 mmol / L, and react at 50° C. for 1 hour. After cooling to room temperature, 50 μL of IPBI with a concentration of 50 mmol / L was added to the above solution, and the reaction was continued at 37° C. for 2 hours. After the reaction was completed, samples were directly sampled for MALDI-TOF MS analysis. The results showed that the derivatization efficiency of the peptide CKDECSLDG was 100% ( figure 1 Middle C panel).

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Abstract

The invention discloses an alkylation derivatization reagent and an application of the alkylation derivatization reagent in peptide fragment marking and mass spectrometric detection. A molecular formula of the alkylation derivatization reagent is brominated 1-[3-[(2-iodine-1-oxo-ethyl) amino] propyl]-3-butyl imidazole (IPBI), and a structure thereof is as shown in Formula (I). The alkylation derivatization reagent takes an iodoacetamide base as an active functional group, and has high hydrophobicity, high gas phase alkalinity and a structural feature of containing permanent positive charges; selective derivatization and efficient mark of active sulfhydryl in polypeptide / protein can be achieved; the charge number of a peptide fragment, the ionization efficiency and the detection sensitivity in mass spectrometric analysis can be increased and improved remarkably, so that a detection rate of a cysteine peptide fragment is increased remarkably; and the alkylation derivatization reagent is simple in synthetic process, lower in cost, good in reaction reproducibility, and convenient to popularize and apply.

Description

technical field [0001] The invention relates to the labeling of biological macromolecules, in particular to an alkylation derivative reagent and its application in peptide labeling and mass spectrometry detection. Background technique [0002] In the post-genomics era, proteome analysis has received more and more attention. However, the extreme complexity of protein sample composition, wide dynamic range distribution, and especially the presence of post-translational modifications make its analysis still face great challenges. The bottom-up strategy represented by the shotgun method is the most commonly used strategy for the separation and identification of complex proteome samples. This strategy first digests the protein sample with protease, and the resulting peptide mixture is separated by chromatography and then passed through mass spectrometry. Perform peptide identification. It can be seen that mass spectrometry is an indispensable tool for proteome research. [000...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D233/61G01N27/62
Inventor 乔晓强王蕊佘丹丹王涛
Owner HEBEI UNIVERSITY
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