Tyrosine phosphoproteomics analysis method of tyrosine phosphopeptide

Abstract

The present invention relates to a method for deep analysis of tyrosine phosphoproteomics based on competitive elution of affinity tyrosine phosphopeptide on a superbinder SH2. Specifically in the elution process of tyrosine phosphopeptide enrichment, a biotin-containing tyrosine phosphopeptide capable of strongly interacting with a superbinder SH2 is used as a competitive reagent to replace the traditional trifluoroacetic acid, the tyrosine phosphopeptide acting with the superbinder SH2 is sequentially eluted according to the intensity of the interaction, and the competitive reagent being notbound to the superbinder SH2 is bound by corresponding avidin so as not to affect the subsequent identification and analysis of the tyrosine phosphopeptide sample. According to the present invention,the method has advantages of rapidness, sensitivity, low cost and the like, can easily achieve the deep analysis of tyrosine phosphoproteomics, and can further provide the information of the interaction between the tyrosine phosphopeptide and the superbinder SH2.

Description

technical field [0001] The invention is a tyrosine phosphorylation proteomics analysis method based on the competitive elution of the affinity tyrosine phosphopeptide on the SH2 superphile. The competition reagent is a tyrosine phosphopeptide segment with biotin, which can be specifically enriched and removed by avidin, and has a strong affinity with the SH2 superphile. During the elution process, the tyrosine phosphopeptides are eluted sequentially according to the affinity with the SH2 superparents, so as to obtain the tyrosine phosphorylated proteome information with the affinity interaction information between the SH2 superparents . If the sequential elution technique is used to further reduce the sample complexity, the number of identified tyrosine phosphopeptides can be increased, the in-depth analysis of the tyrosine phosphoproteome can be achieved, and the interaction between tyrosine phosphopeptides and SH2 superphiles can be obtained at the same time. role informat...

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Problems solved by technology

Due to the severe interference of high-abundance serine / threonine phosphorylation, the analysis of tyrosine phosphopeptides often needs to be achieved by antibody enrichment (Literature 2: Evaluation of different phosphor‐tyrosine antibodies for label‐freephosphoproteomics.J.Proteomics,2015 ,127,259‐263.), but this type of method has disadvantages such as low reproducibility, high price, and poor specificity

Compared with wild-type SH2, the affinity between SH2 superphiles and tyrosine phosphopeptides is greatly improved (Document 3: Superbinder SH2domains act as antagonists of cell signaling. Science Signaling, 2012, 5, ra68‐ra68), and also It has been used to achieve the enrichment analysis of tyrosine phosphopeptides (Document 4: Ultra‐deep tyrosine phosphoproteomics enabled by aphosphotyrosine superbinder. Nat. Chem. Biol., 2016, 12, 959‐966.), but the conventional elution method does not Selective, tyrosine phosphopeptides and other peptides will be eluted together, can only provide a single information, and often need to achieve in-depth analysis of tyrosine phosphorylated proteomes by means of two-dimensional separation

[0003] The above literatures do not record or suggest any in-depth analysis of tyrosine phosphorylated proteomics by using competitive reagents to compete for the elution of the affinity tyrosine phosphopeptides on the SH2 superphile

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