A Strong Cation Exchange Chromatography Protocol for Examining N-Terminal Proteoforms

Methods Mol Biol. 2022:2477:293-309. doi: 10.1007/978-1-0716-2257-5_17.


Especially in eukaryotes, the N-terminal acetylation status of a protein reveals translation initiation sites and substrate specificities and activities of N-terminal acetyltransferases (NATs). Here, we discuss a bottom-up proteomics protocol for the enrichment of N-terminal peptides via strong cation exchange chromatography. This protocol is based on depleting internal tryptic peptides from proteome digests through their retention on strong cation exchangers, leaving N-terminally acetylated/blocked peptides enriched among the nonretained peptides. As such, one can identify novel N-terminal proteoforms and quantify the degree of N-terminal protein acetylation.

Keywords: N-terminal acetylation; N-terminal acetyltransferases; N-terminomics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylation
  • Chromatography
  • Peptides / chemistry
  • Proteome*
  • Proteomics* / methods


  • Peptides
  • Proteome