Protein C-termini study is still a challenging task and far behind its counterpart, N-termini study. MS based C-terminomics study is often hampered by the low ionization efficiency of C-terminal peptides and the lack of efficient enrichment methods. We previously optimized the C-terminal amine-based isotope labeling of substrates (C-TAILS) method and identified 369 genuine protein C-termini in Escherichia coli. A key limitation of C-TAILS is that the prior protection of amines and carboxylic groups at protein level makes Arg-C as the only specific enzyme in practice. Herein, we report an approach combining multi-enzyme digestion and C-TAILS, which significantly increases the identification rate of C-terminal peptides and consequently improves the applicability of C-TAILS in biological studies. We carry out a systematic study and confirm that the omission of the prior amine protection at protein level has a negligible influence and allows the application of multi-enzyme digestion. We successfully apply five different enzyme digestions to C-TAILS, including trypsin, Arg-C, Lys-C, Lys-N, and Lysarginase. As a result, we identify a total of 722 protein C-termini in E. coli, which is at least 66% more than the results using any single enzyme. Moreover, the favored enzyme and enzyme combination are discovered.
[1]Fudan Univ, State Key Lab Genet Engn, Dept Biochem, Sch Life Sci, Shanghai, Peoples R China
(8.0+极速模式)