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Online automatic analysis device and analysis method for phosphorylated proteomics

A phosphorylated protein, automated analysis technology, applied in the fields of chromatography and mass spectrometry, can solve the problems of high back pressure of phosphorylated peptide enrichment column, incompatible loading conditions, poor sensitivity, etc. The effect of improving consistency

Active Publication Date: 2021-11-05
AGRO BIOLOGICAL GENE RES CENT GUANGDONG ACADEMY OF AGRI SCI
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Problems solved by technology

In the actual proteomics analysis tasks, nano-liter liquid chromatography-mass spectrometry is used, but the development of nano-liter phosphopeptide enrichment columns often encounters high back pressure, low sample loading rate, and poor sensitivity Another difficulty is that a high pH solution is required to elute phosphorylated peptides from the capture column, which is incompatible with the loading conditions of the subsequent C18 analytical column. Finally, how to design the connection of the liquid phase system and the automatic analysis program is also a technical problem , so there is no commercially available device for online and automated analysis of phosphorylated proteomes

Method used

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  • Online automatic analysis device and analysis method for phosphorylated proteomics
  • Online automatic analysis device and analysis method for phosphorylated proteomics
  • Online automatic analysis device and analysis method for phosphorylated proteomics

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Embodiment 1

[0051] Example 1 Preparation of ATP-modified immobilized metal ion affinity capillary monolithic column

[0052] The ATP-modified immobilized metal ion affinity capillary monolithic column prepared by a one-step reaction method in the present invention specifically comprises the following steps:

[0053] S1. Add 740 microliters (about 1000 mg) of potassium water glass (modulus 3.3, Baume degree 40) to the micro-reaction flask, and slowly add 6 microliters (about 6 mg) of γ-glycidyl ether oxypropylene under stirring at room temperature Trimethoxysilane (GLYMO, Cas number: 2530-83-8), and continued to stir well at room temperature for 30 minutes.

[0054] S2. Weigh 7.5 mg of adenosine triphosphate disodium, dissolve it in 160 microliters of deionized water, slowly add it to the reaction solution in step S1, and continue to stir fully at room temperature for 30 minutes.

[0055] S3. Take 60 microliters (about 68 mg) of formamide, mix it with 40 microliters of deionized water, sl...

Embodiment 2

[0061] like figure 2 As shown, the online automatic analysis device for phosphoproteomics in this embodiment includes a sample loading part and an analysis part, and the sample loading part includes a sample loading pump 1, a six-way valve 2, and an autosampler 3 (autosampler includes sample disk 13, syringe 14, six-way valve 2 and quantitative loop 11), phosphorylated peptide capture column 4 (i.e. the ATP-modified immobilized metal ion affinity capillary monolithic column of embodiment 1), the The part is mainly responsible for the automated enrichment of phosphorylated peptides. The analysis part includes NC pump 5, C18 pre-column 6, ten-port valve 7, C18 analysis column 8, nano-ESI ion source 9 and high-resolution mass spectrometer 10, which is mainly responsible for online analysis of the enriched phosphorylated peptides. The two ends of the phosphorylated peptide trapping column are respectively connected to the six-port valve and the ten-port valve, and the switching ...

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Abstract

The invention discloses an online automatic analysis device and an analysis method for phosphorylated proteomics. The device is a liquid chromatograph-mass spectrometer, the liquid chromatograph comprises a phosphorylated peptide capture column and an analytical column, and the phosphorylated peptide capture column is an ATP modified immobilized metal ion affinity chromatographic column; and potassium water glass, gamma-glycidyl ether oxypropyl trimethoxy silane and water-dissolved adenosine disodium triphosphate are mixed and stirred, then formamide dissolved with water is added and stirred to obtain a reaction solution, a chromatographic column is filled with the reaction solution, then the reaction solution filling the chromatographic column is subjected to a reaction curing, washing is performed, and the ATP modified immobilized metal ion affinity chromatographic column is obtained. The device has the primary advantage that researchers are liberated from complicated manual operations such as metal ion loading, sample loading, washing, elution, desalting, drying and redissolving required by enrichment of phosphorylated peptides, so that the experiment efficiency is improved.

Description

technical field [0001] The invention belongs to the field of chromatography and mass spectrometry, and in particular relates to an online automatic analysis device and analysis method for phosphorylated proteomics, which perform automatic online analysis of phosphorylated polypeptides on a nanoliter liquid chromatography-mass spectrometer. Background technique [0002] Many important life regulation processes are related to protein phosphorylation. Qualitative and quantitative analysis of protein phosphorylation events in cells will help reveal the regulatory mechanism of phosphorylated modified proteins in signaling pathways, mine key genes or guide drug development. target. Liquid chromatography-high resolution mass spectrometry combined with bottom-up analysis strategy is the core method of current proteomics research. However, due to the extremely low abundance of phosphorylated peptides, poor ionization efficiency, and signal inhibition by non-phosphorylated peptides, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/88
CPCG01N30/88G01N2030/8804
Inventor 晏石娟张文洋黄文洁江彪李文燕吴绍文
Owner AGRO BIOLOGICAL GENE RES CENT GUANGDONG ACADEMY OF AGRI SCI
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