Improved Nα-acetylated peptide enrichment following dimethyl labeling and SCX

Sin Hong Chen, Chiy Rong Chen, Shu Hui Chen, Ding Tzai Li, Jue Liang Hsu

Research output: Contribution to journalArticlepeer-review

20 Citations (Scopus)

Abstract

Protein N-terminal acetylation is one of the most common modifications occurring co- and post-translationally on either eukaryote or prokaryote proteins. However, compared to other protein modifications, the physiological role of protein N-terminal acetylation is relatively unclear. To explore the biological functions of protein N-terminal acetylation, a robust and large-scale method for qualitative and quantitative analysis of this modification is required. Enrichment of Nα-acetylated peptides or depletion of the free N-terminal and internal tryptic peptides prior to analysis by mass spectrometry are necessary based on current technologies. This study demonstrated a simple strong cation exchange (SCX) fractionation method to selectively enrich Nα-acetylated tryptic peptides via dimethyl labeling without the need for tedious protective labeling and depleting procedures. This method was introduced for the comprehensive analysis of N-terminal acetylated proteins from HepG2 cells. Several hundred N-terminal acetylation sites were readily identified in a single SCX flow-through fraction. Moreover, the Nα-acetylated peptides of some protein isoforms were simultaneously observed in the SCX flow-through fraction, which indicated that this approach can be utilized to discriminate protein isoforms with very similar full sequences but different N-terminal sequences, such as β-actin/γ-actin, ERK1/ERK2, α-centractin/β-centractin, and ADP/ATP translocase 2 and 3. Compared to other methods, this method is relatively simple and can be directly implemented in a two-dimensional separation (SCX-RP)-mass spectrometry scheme for quantitative N-terminal proteomics using stable-isotope dimethyl labeling.

Original languageEnglish
Pages (from-to)3277-3287
Number of pages11
JournalJournal of Proteome Research
Volume12
Issue number7
DOIs
Publication statusPublished - 2013 Jul 5

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Chemistry(all)

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