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LysargiNase and Chemical Derivatization Based Strategy for Facilitating In-Depth Profiling of C-Terminome.
Anal Chem 2019AC

Abstract

Global identification of protein C-termini is highly challenging due to their low abundance in conventional shotgun proteomics. Several enrichment strategies have been developed to facilitate the detection of C-terminal peptides. One major issue of previous approaches is the limited C-terminome coverage. Herein, we integrated LysargiNase digestion, chemical acetylation on neo-N-terminus, and a-ion-aided peptide matching into poly(allylamine)-based C-terminomics (termed as LAACTer). In this strategy, we leveraged LysargiNase, a protease with cleavage specificity N-terminal to Lys and Arg residues, to cover previously unidentifiable C-terminome and employed chemical acetylation and a-ion-aided peptide matching to efficiently boost peptide identifications. Triplicates of LAACTer identified a total of 834 C-termini from proteome of 293T cell, which expanded the coverage by 164% (643 more unique C-termini) compared with the parallel experiments using the original workflow. Compared with the largest human C-terminome data sets (containing 800-900 C-termini), LAACTer not only achieved comparable profiling depth but also yielded 465 previously unidentified C-termini. In a SILAC (stable isotope labeling with amino acids in cell culture)-based quantitative study for identification of GluC-cleaved products, LAACTer quantified 300% more C-terminal peptides than the original workflow. Using LAACTer and the original workflow, we performed global analysis for the C-terminal sequences of 293T cell. The original and processed C-termini displayed distinct sequence patterns, implying the "C-end rules" that regulates protein stability could be more complex than just amino acid motifs. In conclusion, we reason LAACTer could be a powerful proteomic tool for in-depth C-terminomics and would benefit better functional understanding of protein C-termini.

Authors+Show Affiliations

State Key Laboratory of Drug Research , Shanghai Institute of Materia Medica, Chinese Academy of Sciences , 555 Zuchongzhi Road , Shanghai , 201203 , China.State Key Laboratory of Drug Research , Shanghai Institute of Materia Medica, Chinese Academy of Sciences , 555 Zuchongzhi Road , Shanghai , 201203 , China. University of Chinese Academy of Sciences , Beijing 100049 , China.State Key Laboratory of Drug Research , Shanghai Institute of Materia Medica, Chinese Academy of Sciences , 555 Zuchongzhi Road , Shanghai , 201203 , China. University of Chinese Academy of Sciences , Beijing 100049 , China.State Key Laboratory of Drug Research , Shanghai Institute of Materia Medica, Chinese Academy of Sciences , 555 Zuchongzhi Road , Shanghai , 201203 , China.State Key Laboratory of Drug Research , Shanghai Institute of Materia Medica, Chinese Academy of Sciences , 555 Zuchongzhi Road , Shanghai , 201203 , China.State Key Laboratory of Drug Research , Shanghai Institute of Materia Medica, Chinese Academy of Sciences , 555 Zuchongzhi Road , Shanghai , 201203 , China.State Key Laboratory of Drug Research , Shanghai Institute of Materia Medica, Chinese Academy of Sciences , 555 Zuchongzhi Road , Shanghai , 201203 , China.State Key Laboratory of Drug Research , Shanghai Institute of Materia Medica, Chinese Academy of Sciences , 555 Zuchongzhi Road , Shanghai , 201203 , China. University of Chinese Academy of Sciences , Beijing 100049 , China.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31634432

Citation

Hu, Hao, et al. "LysargiNase and Chemical Derivatization Based Strategy for Facilitating In-Depth Profiling of C-Terminome." Analytical Chemistry, 2019.
Hu H, Zhao W, Zhu M, et al. LysargiNase and Chemical Derivatization Based Strategy for Facilitating In-Depth Profiling of C-Terminome. Anal Chem. 2019.
Hu, H., Zhao, W., Zhu, M., Zhao, L., Zhai, L., Xu, J. Y., ... Tan, M. (2019). LysargiNase and Chemical Derivatization Based Strategy for Facilitating In-Depth Profiling of C-Terminome. Analytical Chemistry, doi:10.1021/acs.analchem.9b03543.
Hu H, et al. LysargiNase and Chemical Derivatization Based Strategy for Facilitating In-Depth Profiling of C-Terminome. Anal Chem. 2019 Nov 5; PubMed PMID: 31634432.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - LysargiNase and Chemical Derivatization Based Strategy for Facilitating In-Depth Profiling of C-Terminome. AU - Hu,Hao, AU - Zhao,Wensi, AU - Zhu,Mengdi, AU - Zhao,Lei, AU - Zhai,Linhui, AU - Xu,Jun-Yu, AU - Liu,Ping, AU - Tan,Minjia, Y1 - 2019/11/05/ PY - 2019/10/22/pubmed PY - 2019/10/22/medline PY - 2019/10/22/entrez JF - Analytical chemistry JO - Anal. Chem. N2 - Global identification of protein C-termini is highly challenging due to their low abundance in conventional shotgun proteomics. Several enrichment strategies have been developed to facilitate the detection of C-terminal peptides. One major issue of previous approaches is the limited C-terminome coverage. Herein, we integrated LysargiNase digestion, chemical acetylation on neo-N-terminus, and a-ion-aided peptide matching into poly(allylamine)-based C-terminomics (termed as LAACTer). In this strategy, we leveraged LysargiNase, a protease with cleavage specificity N-terminal to Lys and Arg residues, to cover previously unidentifiable C-terminome and employed chemical acetylation and a-ion-aided peptide matching to efficiently boost peptide identifications. Triplicates of LAACTer identified a total of 834 C-termini from proteome of 293T cell, which expanded the coverage by 164% (643 more unique C-termini) compared with the parallel experiments using the original workflow. Compared with the largest human C-terminome data sets (containing 800-900 C-termini), LAACTer not only achieved comparable profiling depth but also yielded 465 previously unidentified C-termini. In a SILAC (stable isotope labeling with amino acids in cell culture)-based quantitative study for identification of GluC-cleaved products, LAACTer quantified 300% more C-terminal peptides than the original workflow. Using LAACTer and the original workflow, we performed global analysis for the C-terminal sequences of 293T cell. The original and processed C-termini displayed distinct sequence patterns, implying the "C-end rules" that regulates protein stability could be more complex than just amino acid motifs. In conclusion, we reason LAACTer could be a powerful proteomic tool for in-depth C-terminomics and would benefit better functional understanding of protein C-termini. SN - 1520-6882 UR - https://www.unboundmedicine.com/medline/citation/31634432/A_LysargiNase-_and_chemical_derivatization-based_strategy_LAACTer_facilitating_in-depth_profiling_of_C-terminome L2 - https://dx.doi.org/10.1021/acs.analchem.9b03543 DB - PRIME DP - Unbound Medicine ER -