A 18o on-line labeled protein quantitative analysis platform and its operation method

Abstract

The invention relates to a <18>O on-line marked protein quantitative analysis platform, and an operation method thereof. The <18>O on-line marked protein quantitative analysis platform is a system integrating protein on-line enzymolysis, <18>O on-line marking and polypeptide separation and detection. The <18>O on-line marked protein quantitative analysis platform comprises an injection pump, a sampling needle, a column oven, an enzyme reactor, a switch valve, a peptide fragment capture column, a liquid phase chromatography pump, a polypeptide separation column and a mass spectrometry detector. A protein sample is firstly loaded to the enzyme reactor through the injection pump to carry out incubation; <18>O marking can be carried out at the same time with enzymolysis; and the <18>O marked peptide fragment after enzymolysis is enriched on-line and transferred into the polypeptide separation column for further separation, and finally is detected by the mass spectrometry detector. The <18>O on-line marked protein quantitative analysis platform integrates the protein on-line enzymolysis, <18>O on-line marking and polypeptide separation.

Description

Technical field: [0001] The present invention relates to a 18 O on-line labeled protein quantitative analysis platform and operation method thereof, 18 O On-line labeled protein quantitative analysis platform is an online enzymatic hydrolysis of proteins, 18 O On-line labeling, peptide separation and detection system. Background technique: [0002] The main separation technique of protein mixture in the initial stage of proteomics is two-dimensional gel electrophoresis (2DE). The protein mixture is fully separated by first-dimensional isoelectric focusing electrophoresis and second-dimensional size exclusion electrophoresis, and then stained by Coomassie brilliant blue or silver. Staining allows the protein separated on the gel to develop color, and the color intensity represents the corresponding protein content in the sample. The relative content of corresponding proteins in the two samples can be obtained by comparing the color intensity of the proteins at the same pos...

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Problems solved by technology

At present, there are mainly two reasons for restricting the further promotion of this method: 1. 18 O marking is less efficient: 18 O labeling is mainly divided into one-step method and two-step method. The one-step method is relatively simple to operate, and only needs to dissolve the sample in 18 O solution can be used for enzymatic hydrolysis, but the labeling efficiency of this method is very low; the labeling efficiency of the two-step method is higher than that of the one-step method, but the operation is cumbersome and time-consuming. 16 O solution for enzymolysis, desalination after enzymolysis, evaporated to dryness, evaporated peptides redissolved in 18 Incubation in a solution of O

2, 18 There is a backcrossing problem with the O mark, which is performed using traditional methods 18 After O labeling, since the protease still exists in the solution, once encountered 16 O solution, marked on 18 The peptide of O will be backcrossed to 16 O (Journal of Proteome Research 2009, 8, 2140-2143; Journal of Proteome Research 2009, 8, 2157-2163)

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