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

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 (P =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 (P =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.