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An Approach to Incorporate Multi‐Enzyme Digestion into C‐TAILS for C‐Terminomics Studies
Author(s) -
Zhang Yang,
Li Qingqing,
Huang Jingnan,
Wu Zhen,
Huang Jichang,
Huang Lin,
Li Yanhong,
Ye Juanying,
Zhang Xumin
Publication year - 2018
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201700034
Subject(s) - enzyme , chemistry , trypsin , biochemistry , escherichia coli , digestion (alchemy) , amine gas treating , computational biology , biology , chromatography , gene , organic chemistry
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. Data are available via ProteomeXchange with identifier PXD004275.