Systematic optimization of C-TAILS approach for deep screening of C-terminomics

ABSTRACT: 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 structure and understanding protein functions. Although many efforts have been made in the fields during the latest two decades, the progress is still far behind its counterpart, N-termini, and it necessitates more novel or optimized methods. Herein, we reported 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 E. Coli sample, about 42% higher than the conventional C-TAILS. A systematic data analysis revealed that the optimized method is unbiased to amino acid composition, more reproducible and with higher MASCOT scores. Moreover, the introduction of SCII (Single-Charge Ion Inclusion) 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 μg each. Our optimized method would benefit the deep screening of C-terminomics and possibly help discover some novel C-terminal modifications.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Escherichia Coli

TISSUE(S): Whole Body

SUBMITTER: yang zhang  

LAB HEAD: xumin zhang

PROVIDER: PXD002409 | Pride | 2015-09-15

REPOSITORIES: Pride