内源性蛋白质交联的生物质谱分析进展

Endogenous protein crosslinking usually means the connection of two amino acids in one or two proteins by covalent bonds, which includes intermolecular and intramolecular crosslinks. As a special posttranslational modification of proteins, endogenous protein crosslinking affects the structure and function of the proteins. The main endogenous protein crosslinks currently discovered in prokaryotes and eukaryotes are disulfide bonds, isopeptide bonds, ditryptophan, dityrosine, formaldehyde, methylglyoxal, NOS bridges and so on. All these crosslinks have been shown to have important effects on biological processes and molecular functions. The formation of endogenous protein crosslinking comprises the enzymecatalyzed and non-enzyme-catalyzed. It is reported that several enzymes are utilized to crosslink the proteins in the literature, such as protein disulfide isomerase, transglutaminase, sortase A, tyrosinase, laccase and peroxidase. Some enzymes are already applied for protein crosslinking in the field of the food industry. However, the endogenous substrates of these enzymes are not fully discovered in vivo so far. In addition, the mechanism of protein crosslinks and the function of crosslink protein require further exploration. Mass spectrometry is a high-throughput and high-sensitivity tool that is reliable for the large-scale identification of endogenous protein crosslinking. In recent years, there have been an everincreasing number of studies on endogenous protein crosslinking by mass spectrometry at the omics level, and mass spectrometry has become one important tool to study the endogenous protein crosslinking. For example, it is reported that for the first time, 199 disulfide bonds are identified in Escherichia coli and 568 disulfide bonds are identified in secreted proteins of endothelial cells. The results show that disulfide bonds can be divided into two categories: structural and allosteric disulfide bonds. Allosteric disulfide bonds are involved in catalysis and regulation in the cell. Besides the disulfide bond, other types of endogenous protein crosslinking also need to be identified and quantified, which will better understand their functions. With the development of mass spectrometry technology and identification methods, the study of endogenous protein crosslinking will be enhanced to a deeper level. From the early stage, the crosslinking can be identified from single purified protein, followed by the standard protein mixtures, and now, large-scale identification can be achieved in complex samples, such as in the prokaryotic and eukaryotic cells. However, there are still many challenges in the process of sample preparation, data acquisition, qualitative and quantitative analysis and bioinformatics software. It is urgent to establish a unique workflow to study the endogenous protein crosslinking technology, which will greatly promote the development of crosslinking omics. In the present paper, we summarize the important achievements in the recent studies of endogenous protein crosslinking, sort out problems on endogenous protein crosslinking analysis based on mass spectrometry, and finally put forward the prospect of developing endogenous protein crosslinking at the omics level.