title: Depletion of internal peptides by site-selective blocking, phosphate labeling, and TiO2 adsorption for in-depth analysis of C-terminome.
abstract: The analysis of protein C-termini is of great importance, because it not only provides valuable information about protein function, but also facilitates the elucidation of proteolytic processing. However, even with the recent methods for the global profiling of protein C-termini, the identification of C-termini is still far behind that of N-termini due to the lack of basic residue and low reactive carboxyl group. Therefore, an unbiased and complementary method for C-termini profiling is imperative. In this work, we developed a negative enrichment strategy to achieve the in-depth analysis of C-terminome. Proteins were firstly amidated to block carboxyl groups, followed by lysyl endoproteinase (LysC) digestion to generate C-terminal peptides with α-amines and internal peptides bearing both α- and ε-amines. After the α-amines were blocked by site-selective dimethylation or succinylation, the remaining ε-amines on internal peptides were labeled with phosphate groups. Finally, internal peptides were depleted by TiO2, leaving exclusively the fraction of C-terminal peptides for LC-MS/MS analysis. With Escherichia coli (E. coli) digests as the sample, the efficiency of amidation, dimethylation/succinylation, phosphate labeling and TiO2 depletion was proved high. With the combination of dimethyl and succinic blocking strategy, our method enabled the identification of 477 unique C-terminal peptides in E. coli. In comparison with the C-terminal amine-based isotope labeling of substrates (C-TAILS) method, 83 C-termini were identified by both methods, whereas 369 C-termini were unique to C-TAILS and 394 to our dataset. The method proposed is therefore efficient and possibly promotes the comprehensive profiling of C-termini. Graphical Abstract Negative isolation of C-terminal peptides with combination of site-selective blocking, phosphate labeling, and TiO2 adsorption.
authors.affiliation: National Chromatographic R. & A. Center, Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, Liaoning, 116023, China.|University of Chinese Academy of Sciences, Beijing, 100049, China.
authors.forename: Lingfan
authors.initials: L
authors.lastname: Chen
authors.affiliation: National Chromatographic R. & A. Center, Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, Liaoning, 116023, China.
authors.forename: Yichu
authors.initials: Y
authors.lastname: Shan
authors.affiliation: National Chromatographic R. & A. Center, Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, Liaoning, 116023, China.|University of Chinese Academy of Sciences, Beijing, 100049, China.
authors.forename: Yejing
authors.initials: Y
authors.lastname: Weng
authors.affiliation: National Chromatographic R. & A. Center, Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, Liaoning, 116023, China.
authors.forename: Huiming
authors.initials: H
authors.lastname: Yuan
authors.affiliation: National Chromatographic R. & A. Center, Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, Liaoning, 116023, China.|University of Chinese Academy of Sciences, Beijing, 100049, China.
authors.forename: Shen
authors.initials: S
authors.lastname: Zhang
authors.affiliation: The Beijing SHRIMP Center, Institute of Geology, Chinese Academy of Geological Sciences, Beijing, 102206, China.
authors.forename: Runlong
authors.initials: R
authors.lastname: Fan
authors.affiliation: National Chromatographic R. & A. Center, Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, Liaoning, 116023, China.
authors.forename: Zhigang
authors.initials: Z
authors.lastname: Sui
authors.affiliation: National Chromatographic R. & A. Center, Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, Liaoning, 116023, China.
authors.forename: Xiaodan
authors.initials: X
authors.lastname: Zhang
authors.affiliation: National Chromatographic R. & A. Center, Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, Liaoning, 116023, China. lihuazhang@dicp.ac.cn.
authors.forename: Lihua
authors.initials: L
authors.lastname: Zhang
authors.affiliation: National Chromatographic R. & A. Center, Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, Liaoning, 116023, China.
authors.forename: Yukui
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000327:Adsorption; D002853:Chromatography, Liquid; D010455:Peptides / Q000737:chemistry*; D010710:Phosphates / Q000737:chemistry*; D053719:Tandem Mass Spectrometry; D014025:Titanium / Q000737:chemistry*
chemical_list: D010455:Peptides; D010710:Phosphates; C009495:titanium dioxide; D014025:Titanium
keywords: C-termini; Dimethylation; Proteolysis; Succinylation; TiO2 particle