title: Systematic Optimization of C-Terminal Amine-Based Isotope Labeling of Substrates Approach for Deep Screening of C-Terminome.
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 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 μg 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.
authors.affiliation: State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University , Shanghai 200438, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Center for Reproduction and Genetics, Suzhou Municipal Hospital, Nanjing Medical University Affiliated Suzhou Hospital , Suzhou, Jiangsu 215002, China.
authors.forename: Quanze
authors.initials: Q
authors.lastname: He
authors.affiliation: State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University , Shanghai 200438, China.
authors.forename: Juanying
authors.initials: J
authors.lastname: Ye
authors.affiliation: State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University , Shanghai 200438, China.
authors.forename: Yanhong
authors.initials: Y
authors.lastname: Li
authors.affiliation: State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University , Shanghai 200438, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University , Shanghai 200438, China.
authors.forename: Qingqing
authors.initials: Q
authors.lastname: Li
authors.affiliation: State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University , Shanghai 200438, China.
authors.forename: Jingnan
authors.initials: J
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University , Shanghai 200438, China.
authors.forename: Jianan
authors.initials: J
authors.lastname: Lu
authors.affiliation: State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University , Shanghai 200438, China.
authors.forename: Xumin
authors.initials: X
authors.lastname: Zhang
mesh_terms: D000588:Amines / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry; D007553:Isotope Labeling; D010455:Peptides / Q000032:analysis* / Q000737:chemistry*
chemical_list: D000588:Amines; D010455:Peptides