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

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 charge sat 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 to20 μg of human HEK293T cell lysate proteins to profile theN-terminal proteome. On average, 1550 acetylated and200 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 proteo forms with neo-Ntermini.
Original languageEnglish
Article number100003
Number of pages9
JournalMolecular and Cellular Proteomics
Volume20
Early online date23 Nov 2020
DOIs
Publication statusPublished - 1 Jan 2021

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