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Method for enhancing ionization efficiency of peptide segment

An ionization and high-efficiency technology, applied in the field of biochemical analysis, can solve the problems of poor mass spectrometry detection, difficult removal of high-abundance protein suppression, increased sample loss, pollution, etc., to improve ionization efficiency and increase mass spectrometry detection sensitivity , Improve the effect of ionization efficiency

Inactive Publication Date: 2011-07-27
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the specificity of these methods is not high enough, the steps are cumbersome and time-consuming, which greatly increases the possibility of sample loss and contamination, and is not suitable for high-throughput protein identification
For low-abundance proteins, enrichment is still the most important method, and this method requires a large number of samples to support, and the suppression of high-abundance proteins is also difficult to remove, resulting in the mass spectrometry detection of them is still not very good improvement

Method used

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  • Method for enhancing ionization efficiency of peptide segment
  • Method for enhancing ionization efficiency of peptide segment
  • Method for enhancing ionization efficiency of peptide segment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 verifies that the reaction efficiency of the derivatization reaction is close to 100%

[0031] 1-(2-pyrimidinyl)piperazine was diluted in N,N-dimethylformamide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1 -Hydroxy-7-azobenzotriazole was respectively dissolved in N,N-dimethylformamide to prepare a solution of 2 mg / ml. Add 6, 4 and 3 microliters of the above three reagents in sequence to 50 microliters of peptide KRGSGAW aqueous solution with a concentration of about 100 nanograms per microliter, and adjust the pH value to between 7.5 and 7.8 with 0.1% trifluoroacetic acid aqueous solution between. The above mixed system was suspended at room temperature for 30 seconds. It was then dried by centrifugation under vacuum to remove all solvents. The above-mentioned dried product was dissolved in an aqueous solution containing 50% acetonitrile and 0.1% trifluoroacetic acid for MALDI-MS detection, and the results showed that the derivatization...

Embodiment 2

[0032] Example 2 verifies that the reaction efficiency of the derivatization reaction is close to 100%

[0033] 1-(2-pyrimidinyl)piperazine was diluted in N,N-dimethylformamide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1 -Hydroxy-7-azobenzotriazole was respectively dissolved in N,N-dimethylformamide to prepare a solution of 2 mg / ml. Add 6, 4 and 3 microliters of the above three reagents in sequence to 50 microliters of peptide DRVYIHPF aqueous solution with a concentration of about 100 nanograms per microliter, and adjust the pH value to between 7.5 and 7.8 with 0.1% trifluoroacetic acid aqueous solution between. The above mixed system was suspended at room temperature for 30 seconds. It was then dried by centrifugation under vacuum to remove all solvents. The above dried product was dissolved in an aqueous solution containing 50% acetonitrile and 0.1% trifluoroacetic acid for MALDI-MS detection, and the efficiency of the two-site derivatization react...

Embodiment 3

[0034] Example 3 The multiple of the ionization efficiency of the peptide segment RPGFSP after derivatization

[0035] 1-(2-pyrimidinyl)piperazine was diluted in N,N-dimethylformamide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1 -Hydroxy-7-azobenzotriazole was respectively dissolved in N,N-dimethylformamide to prepare a solution of 2 mg / ml. Add 6, 4 and 3 microliters of the above three reagents in sequence to 50 microliters of the peptide RPGGFSP aqueous solution with a concentration of about 100 nanograms per microliter, and adjust the pH value to between 7.5 and 7.8 with 0.1% trifluoroacetic acid aqueous solution between. The above mixed system was suspended at room temperature for 30 seconds. It was then dried by centrifugation under vacuum to remove all solvents. Add the same amount of underivatized peptides to each peptide derivative product, and dissolve the above products with an aqueous solution containing 50% acetonitrile and 0.1% trifluoroace...

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Abstract

The invention belongs to the biochemical analysis field and relates to a method for improving the peptide segment ionization efficiency. An organic compound with alkalescence and hydrophobic nature is utilized to conduct derivation for protein or peptide segment carboxyl, so as to improve the peptide segment ionization efficiency. All derived samples perform mass spectrometric analysis and identification directly. The organic compound selected in the invention can form a covalent bond with the peptide segment carboxyl with high efficiency and high specificity, thereby effectively improving the combining capacity of the peptide segment and protons, greatly improving the peptide segment ionization efficiency, and enabling the peptide segment with low abundance ratio and difficult ionizationto be detected. The invention has the advantages of high reaction specialty, few secondary reaction, simple steps, economy, time-saving, and the like. The invention can be widely used in the proteomics field, and greatly improve the detection capacity of mass spectrum for the peptide segment with low abundance ratio and difficult ionization.

Description

technical field [0001] The invention belongs to the field of biochemical analysis and relates to a method for improving the ionization efficiency of peptide segments. It specifically involves the use of basic and hydrophobic organic compounds to derivatize the carboxyl groups of polypeptide and protein samples, so that the ionization efficiency of mass spectrometry is improved, and the derivatized samples are directly analyzed and identified by mass spectrometry. Background technique [0002] With the implementation and advancement of the Human Genome Project (HGP), life science research has entered the post-genome era. At this stage, the main research object of life science is functional genomics, including structural genomics research and proteomics research. The task of elucidating the expression rules and biological functions of all the proteins expressed in the genome that actually perform life activities is more complex and arduous than elucidating the rules of gene a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N1/28G01N27/64
Inventor 许亚伟张莉娟陆豪杰杨芃原
Owner FUDAN UNIV
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