Combination of multi-enzyme digestions and C-TAILS approach for deep screening of C-terminome

ABSTRACT: Protein C-termini study is still a challenging task and far behind its counterpart N-termini study. The identification of C-termini is often hampered by low ionization response in MS and fewer efficient enrichment methods existing. We previously optimized the C-TAILS (C-terminal amine-based isotope labeling of substrates) method to achieve 75% more identification of protein C-terminal peptides. Although with the significant improvement, the performance of method is not comparable with a regular proteome study since the identification number is very limited. Herein we reported an optimized method by introduction of multiple-enzyme digestions in order to increase the capacity and application of C-TAILS in biological and clinical study. To allow the application of other enzymes than Arg-C in C-TAILS method, we evaluated the effect of the omission of prior amine protection at protein level and revealed the negligible effect caused by. Subsequently we applied five different enzymes in the C-TAILS and evaluated the different performances on the basis of identification and complementariness. As a result, 722 protein C-termini of E. coli were identified in a triplicate experiments using 50 μg each, and the favored enzyme and enzyme combination were discovered and discussed.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Escherichia Coli

TISSUE(S): Whole Body

SUBMITTER: yang zhang  

LAB HEAD: Xumin Zhang

PROVIDER: PXD004275 | Pride | 2017-11-24

REPOSITORIES: Pride