LAUSR

Protein N-termini and their modifications not only represent different protein isoforms, but also relate to the functional annotation and proteolytic activities. Currently, negative selection methods, such as terminal amine isotopic labeling of substrates (TAILS), are the most popular strategy to analyze the protein N-terminome, in which dimethylation or acetylation modification is commonly used to block the free amines of proteome samples. However, after tryptic digestion, the generated long peptides, caused by the missing cleavage of blocked lysine, could hardly be identified by MS, which hindered the deep-coverage analysis of N-terminome. Herein, to solve this problem, we developed an approach, named terminal amine guanidination of substrates (TAGS). 1H-Pyrazole-1-carboxamidine was used to effectively guanidinate lysine ε-amine and N-terminal α-amine, followed by tryptic digestion to generate N-terminal peptides without free amines and internal peptides with free amines. Then, the internal peptides with free amines were removed by hyperbranched polyglycerol-aldehyde polymers (HPG-ALDs), to achieve the negative enrichment of N-terminome. By TAGS, not only the cleavage rate of blocked lysine could be improved, but also the ionization efficiency of tryptic peptides was increased. By comparison, 1814 and 1620 protein N-termini were respectively identified by TAGS and TAILS in S. cerevisiae. Among them, 1012 N-termini were uniquely identified in TAGS. Furthermore, by the combination of TAGS and SILAC of label-free quantitative method, we not only identified the known N-terminal cleavage fragment of gasdermin-D, but also identified some new cleavage sites during Val-boroPro-induced pyroptosis. All these results demonstrated that our developed approach, TAGS, might be of great promising for the comprehensive analysis of N-terminome, beneficial to promote the identification of protein isoforms and the in-depth study the proteolytic activity of proteins.