Method for identifying ubiquitin-like modification sites of proteins

Abstract

The invention relates to a method for identifying the ubiquitin-like modification sites of proteins. The above specific enrichment labeling identification method comprises the following steps: carrying out enzymatic hydrolysis of purified ubiquitin-like modification proteins, enriching the ubiquitin-like modification enzyme digestion peptide fragments through a specific ubiquitin-like antibody, protecting the non-modification lysine amino residues in the enriched peptide fragments through utilizing chemical labeling, carrying out a deubiquinaning enzyme catalysis reaction to expose modified lysine on a substrate peptide fragment sequence, analyzing the peptide segment sequences through a liquid chromatograph-mass spectrometer, and matching through protein mass spectrum data analysis software to obtain the chemically modified peptide fragment sequence, wherein lysine sites containing free amino groups in the finally obtained sequence are the ubiquitin-like modification sites. Compared with common gene mutation methods for obtaining the sites at present, the method provided by the invention has an advantage that information about the ubiquitin-like modification sites of proteins can be rapidly, efficiently and accurately obtained without a tedious gene mutation technology.

Description

technical field [0001] The invention relates to the field of proteomics, more specifically, the identification and application of protein ubiquitination modification sites. Background technique [0002] There are a variety of protein post-translational modifications in cells, such as phosphorylation, acetylation, ubiquitination, methylation, etc., and they all play an important role in the normal biological functions of proteins. Among them, ubiquitylation is one of the important ways to mediate protein degradation. By binding 76 amino acids of ubiquitin (Ub) to target proteins to form polyubiquitin chains, they are recognized by proteasomes and cause protein degradation. degradation. In recent years, several ubiquitin-like proteins (ubiquitin-like proteins, Ubls) have been discovered one after another. Their structures are similar to ubiquitin, and they have a wide range of functions in cells. SUMO (small ubiquitin-related modifier) ​​is one of the important members. SUMO...

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Problems solved by technology

The main reasons are as follows: First, due to the dynamic and reversible nature of SUMOylation, the proportion of SUMOylated proteins in normal cells does not exceed 5% of the total protein, and many proteins can only be stimulated under specific conditions such as cell cycle, nutritional status, Heat shock, DNA damage, etc. will be modified by SUMOylation. How to enrich enough SUMOylated peptides for identification and eliminate the interference of unmodified peptides has always been a research difficulty; The molecular weight of the SUMO-modified peptide after enzyme digestion has a significant shift (SUMO-1 modification increases the molecular weight of the peptide by 2136Da, and SUMO-2 / 3 modification increases the molecular weight of the peptide by 3552Da). If the ratio is not within the detection range of mass spectrometry (400-1800), it cannot be selected for MS / MS sequence identification; third, when high molecular weight SUMOylated peptides are selected for collision-induced dissociation in primary mass spectrometry, due to Collision energy is limited, and often cannot provide sufficient fragment ions for sequence identification; fourth, unlike phosphorylation, the amino acid sequence of the SUMO modification will also undergo collision dissociation, making the entire SUMOylated peptide MS / MS The mass spectrum is quite complex

Despite attempts, there is currently no commercially available data analysis software that can perform high-throughput identification of these SUMO-modified MS / MS spectra to identify substrate peptide sequences and modification sites

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