Isolation of acetylated and unmodified protein n-terminal peptides by strong cation exchange chromatographic separation of trypn-digested peptides

Chih Hsiang Chang, Hsin Yi Chang, Juri Rappsilber, Yasushi Ishihama

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)

Abstract

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.

Original languageEnglish
Article number002148
JournalMolecular and Cellular Proteomics
Volume20
DOIs
Publication statusPublished - 2021

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Molecular Biology

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