An online automated analysis device and analysis method for phosphoproteomics

Abstract

The invention discloses an online automatic analysis device and analysis method for phosphorylated proteomics. It is a liquid chromatography-mass spectrometer, and the liquid chromatography includes a phosphorylated peptide capture column and an analysis column, and the phosphorylated peptide capture column is an ATP-modified immobilized metal ion affinity chromatography column; the potassium Mix and stir water glass, γ-glycidyl etheroxypropyl trimethoxysilane and water-dissolved adenosine triphosphate disodium, then add formamide dissolved in water and stir to obtain a reaction solution, fill the reaction solution with the chromatographic column, and then fill the chromatographic column The reaction solution of the column is solidified by reaction, and then washed to obtain an ATP-modified immobilized metal ion affinity chromatography column. The primary advantage of the present invention is that researchers are freed from complex manual operations such as metal ion loading, sample loading, washing, elution, desalination, evaporation, and reconstitution required for enrichment of phosphorylated peptides, which improves experimental efficiency .

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, ...

Examples

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] Such as 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 switchi...

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, ...

Examples

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] Such as 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 switchi...

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