LAUSR

Global identification of protein C-termini is highly challenging due to their low abundance in conventional shotgun proteomics. Several enrichment strategies has been developed to facilitate the detection of C-terminal peptides. One major issue of previous approaches is the limited C-terminome coverage. Herein, we integrated LysargiNase digestion, chemical acetylation on neo-N-terminus, and a-ion-aided peptide matching into polyallylamine-based C-terminomics (termed as LAACTer). In this strategy, we leveraged a less-exploited protease, LysargiNase, to cover previously unidentifiable C-terminome, and employed chemical acetylation and a-ion-aided peptide matching to efficiently boost peptide identifications. Triplicates of LAACTer identified a total of 834 C-termini from proteome of 293T cell, which expanded the coverage by 164% (643 more unique C-termini) compared with the parallel experiments using original workflow. Compared with the largest human C-terminome datasets (containing 800~900 C-termini), LAACTer not only achieved comparable profiling depth but also yielded 465 previously unidentified C-termini. When coupled with the stable-isotope labelling by amino acids in cell culture (SILAC) technology to identify the GluC-cleaved products, LAACTer quantified 400% more C-terminal peptides than the original workflow. By in-depth C-terminome profiling, we globally analyzed the sequence features of both original and truncated C-termini. From the results, we suggested LAACTer could be useful for analyses of cellular proteolysis events and the C-terminus-related regulatory mechanism of protein stability. In conclusion, we reason LAACTer could be a powerful proteomic tool for in-depth C-terminomics and would benefit better functional understanding of protein C-termini.