Enhanced methylarginine characterization by post-translational modification-specific targeted data acquisition and electron-transfer dissociation mass spectrometry

Gene Hart-Smith, Jason K. K. Low, Melissa A. Erce, Marc R. Wilkins*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

30 Citations (Scopus)

Abstract

When localizing protein post-translational modifications (PTMs) using liquid-chromatography (LC)- tandem mass spectrometry (MS/MS), existing implementations are limited by inefficient selection of PTM-carrying peptides for MS/MS, particularly when PTM site occupancy is sub-stoichiometric. The present contribution describes amethod by which peptides carrying specific PTMs of interest-in this study, methylarginines-may be selectively targeted for MS/MS: peptide features are extracted from high mass accuracy single-stage MS data, searched against theoretical PTM-carrying peptide masses, and matching features are subjected to targeted data acquisition LC-MS/MS. Using trypsin digested Saccharomyces cerevisiae Npl3, in which evidence is presented for 18 methylarginine sites- 17 of which fall within a glycine-arginine-rich (GAR) domain spanning <120 amino acids-it is shown that this approach outperforms conventional data dependent acquisition (DDA): when applied to a complex protein mixture featuring in vivo methylated Npl3, 95 % more (=0.030) methylargininecarrying peptides are selected for MS/MS than DDA, leading to an 86 % increase (P =0.044) in the number of methylated peptides producing Mascot ion scores ≥20 following electron-transfer dissociation (ETD). Notably, significantly more low abundance arginine methylated peptides (maximum ion intensities <6×104 cps) are selected for MS/MS using this approach relative to DDA (50 % more in a digest of purified in vitro methylated Npl3). It is also demonstrated that relative to collision-induced dissociation (CID), ETD facilitates a 586 % increase (=0.016) in average Mascot ion scores of methylarginine-carrying peptides. The present PTM-specific targeted data acquisition approach, though described using methylarginine, is applicable to any ionizable PTMof known mass.

Original languageEnglish
Pages (from-to)1376-1389
Number of pages14
JournalJournal of the American Society for Mass Spectrometry
Volume23
Issue number8
DOIs
Publication statusPublished - Aug 2012
Externally publishedYes

Keywords

  • Post-translational modification
  • Methylarginine
  • Shotgun proteomics
  • Electron-transfer dissociation
  • Saccharomyces cerevisiae
  • MRNA-binding protein Npl3

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