Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

A novel LysargiNase- and chemical derivatization-based strategy LAACTer greatly facilitating in-depth profiling of C-terminome (#577)

Hao Hu 1 , Wensi Zao 1 , Mengdi Zhu 1 , Linhui Zhai 1 , Minjia Tan 1
  1. Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Pudong, SHANGHAI, China

Protein C-termini impact protein functions in multifaceted way, including localization, stability, and activity, etc. Among proteome-wide C-termini enrichment strategies, polyallylamine enrichment-based C-terminomics (PECTer), such as C-TAILS, represents a promising one due to the relatively simple workflow. Nonetheless, it still suffers from trypsin-related sequence bias and low identification rates. We sought to leverage the N-terminal cleavage specificity of LysargiNase to address these issues. By incorporation of LysargiNase digestion, neo-N-terminal acetylation, and a-ion-aided peptide matching into PECTer (termed LAACTer), we achieved to increase the average identification number by ~150% and total coverage by ~160%. LAACTer, together with PECTer, identified a total of ~ 1000 unique C-termini, which represented the most prominent improvement of PECTer so far. As to the quantitative performance, LAACTer showed even higher orthogonality to PECTer as revealed by identification for GluC cleavage products where only 2% of the total peptides were commonly identified by two methods. Finally, by combinational use of LAACTer and the PECTer, we revealed several novel functional insights for the C-termini of 293T cell, including sequence features related to protein stability, function alteration of proteins due to C-terminal processing, and possible truncation mechanisms. In conclusion, the newly established LAACTer will assist to deep C-terminome profiling and find wide applications in exploration for biological roles of protein C-termini.