TY - JOUR
T1 - Isolation of acetylated and unmodified protein n-terminal peptides by strong cation exchange chromatographic separation of trypn-digested peptides
AU - Chang, Chih Hsiang
AU - Chang, Hsin Yi
AU - Rappsilber, Juri
AU - Ishihama, Yasushi
N1 - Funding Information:
ported by JST Strategic Basic Research Program CREST (No. 18070870), AMED Advanced Research and Development Programs for Medical Innovation CREST (18068699), and JSPS Grant-in-Aid for Scientific Research No. 17H05667 (to Y. I.), the Wellcome Trust No. 103139 (to J. R.) and JSPS Invitational Fellowship for Research in Japan No. L16568 (to J. R. and Y. I.). The Wellcome Centre for Cell Biology is supported by core funding from the Wellcome Trust (No. 203149).
Publisher Copyright:
© 2020 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology.
PY - 2021
Y1 - 2021
N2 - We developed a simple and rapid method to enrich protein N-terminal peptides, in which the protease TrypN is first employed to generate protein N-terminal peptides without Lys or Arg and internal peptides with two positive charges at their N termini, and then, the N-terminal peptides with or without N-acetylation are separated from the internal peptides by strong cation exchange chromatography according to a retention model based on the charge/ orientation of peptides. This approach was applied to 20 μg of human HEK293T cell lysate proteins to profile the N-terminal proteome. On average, 1550 acetylated and 200 unmodified protein N-terminal peptides were successfully identified in a single LC/MS/MS run with less than 3% contamination with internal peptides, even when we accepted only canonical protein N termini registered in the Swiss-Prot database. Because this method involves only two steps, protein digestion and chromatographic separation, without the need for tedious chemical reactions, it should be useful for comprehensive profiling of protein N termini, including proteoforms with neo-N termini.
AB - We developed a simple and rapid method to enrich protein N-terminal peptides, in which the protease TrypN is first employed to generate protein N-terminal peptides without Lys or Arg and internal peptides with two positive charges at their N termini, and then, the N-terminal peptides with or without N-acetylation are separated from the internal peptides by strong cation exchange chromatography according to a retention model based on the charge/ orientation of peptides. This approach was applied to 20 μg of human HEK293T cell lysate proteins to profile the N-terminal proteome. On average, 1550 acetylated and 200 unmodified protein N-terminal peptides were successfully identified in a single LC/MS/MS run with less than 3% contamination with internal peptides, even when we accepted only canonical protein N termini registered in the Swiss-Prot database. Because this method involves only two steps, protein digestion and chromatographic separation, without the need for tedious chemical reactions, it should be useful for comprehensive profiling of protein N termini, including proteoforms with neo-N termini.
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U2 - 10.1074/MCP.TIR120.002148
DO - 10.1074/MCP.TIR120.002148
M3 - Article
C2 - 33139343
AN - SCOPUS:85101293548
SN - 1535-9476
VL - 20
JO - Molecular and Cellular Proteomics
JF - Molecular and Cellular Proteomics
M1 - 002148
ER -