Protein ubiquitination modification site detection method based on high-precision mass spectrum and application

Abstract

The invention discloses a protein ubiquitination modification site detection method based on a high-precision mass spectrum and application, and the protein ubiquitination modification site detection method comprises the following steps: S1, constructing an in-vitro SUMOylation modification reaction system to obtain a target protein; S2, carrying out trypsin digestion on the target protein obtained in the step S1, so as to obtain a polypeptide fragment after enzyme digestion; S3, performing liquid chromatography-mass spectrometry analysis on the polypeptide obtained in the step S2 to acquire polypeptide mass spectrum data, and performing data analysis to obtain a sequence of a modification site and a core peptide fragment; S4, chemically synthesizing an isotope labeled peptide fragment sequence containing SUMOylation modification sites, and carrying out site confirmation by adopting a PRM method to determine a set of parent-daughter ion pairs; S5, detecting an endogenous modification site by using the parent-daughter ion pair established in the step S4; the SUMOylation modification refers to ubiquitination modification of the SUMO1 type.

Description

technical field [0001] The invention relates to the detection of protein post-translation modification sites, more specifically, the detection and application of protein ubiquitination modification sites. Background technique [0002] Protein post-translational modifications (PTMs) are an important way to regulate protein function, which is crucial to the structure and function of proteins under physiological and pathological conditions. Specifically, it refers to the process in which a specific position of the amino acid sequence of a protein is covalently bonded to a chemical group or a small molecule protein during the process from the initiation of translation to the formation of a functional protein, and the molecular weight of the protein is changed by adding or subtracting specific modifying groups , hydrophobicity, conformation and stability to affect the function of the protein, these modifications include phosphorylation, glycosylation, ubiquitination, acetylation,...

AI Technical Summary

Problems solved by technology

However, the technical solution disclosed in this patent document has the following defects: 1. Only based on double arsenic dye labeling, single-molecule imaging technology is used to analyze whether the protein to be detected undergoes ubiquitin-like modification, but the amino acid that undergoes ubiquitin-like modification cannot be clearly identified site

2. Since the protein to be detected is transferred from an exogenous source, it cannot truly reflect the endogenous modification level

Even when quantified, only the levels of monoubiquitination and polyubiquitinatio

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