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-amines and N-terminal α-amines, 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.
In comparison, 1814 and 1620 protein N-termini were, respectively,
identified by TAGS and TAILS in Saccharomyces cerevisiae (S. cerevisiae). Among them, 1012 N-termini
were uniquely identified in TAGS. Furthermore, by the combination
of TAGS and the stable isotope labeling with amino acids in cell culture
(SILAC)/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 promise for the comprehensive analysis of N-terminome
and beneficial for promoting the identification of protein isoforms
and studying in-depth the proteolytic activity of proteins.