It is well-known that protein C-termini play important roles in various biological processes, and thus the precise characterization of C-termini is essential for fully elucidating protein structures and understanding protein functions. Although many efforts have been made in the field during the latest 2 decades, the progress is still far behind its counterpart, N-termini, and it necessitates more novel or optimized methods. Herein, we report an optimized C-termini identification approach based on the C-terminal amine-based isotope labeling of substrates (C-TAILS) method. We optimized the amidation reaction conditions to achieve higher yield of fully amidated product. We evaluated different carboxyl and amine blocking reagents and found the superior performance of Ac-NHS and ethanolamine. Replacement of dimethylation with acetylation for Lys blocking resulted in the identification of 232 C-terminal peptides in an Escherichia coli sample, about 42% higher than the conventional C-TAILS. A systematic data analysis revealed that the optimized method is unbiased to the number of lysine in peptides, more reproducible and with higher MASCOT scores. Moreover, the introduction of the Single-Charge Ion Inclusion (SCII) method to alleviate the charge deficiency of small peptides allowed an additional 26% increase in identification number. With the optimized method, we identified 481 C-terminal peptides corresponding to 369 C-termini in E. coli in a triplicate experiments using 80 mu g each. Our optimized method would benefit the deep screening of C-terminome and possibly help discover some novel C-terminal modifications. Data are available via ProteomeXchange with identifier PXD002409.
[1]Fudan Univ, Sch Life Sci, Dept Biochem, State Key Lab Genet Engn, Shanghai 200438, Peoples R China.
[2]Nanjing Med Univ, Affiliated Suzhou Hosp, Suzhou Municipal Hosp, Ctr Reprod & Genet, Suzhou 215002, Jiangsu, Peoples R China.
(8.0+极速模式)