title: Large Scale Mass Spectrometry-based Identifications of Enzyme-mediated Protein Methylation Are Subject to High False Discovery Rates.
abstract: All large scale LC-MS/MS post-translational methylation site discovery experiments require methylpeptide spectrum matches (methyl-PSMs) to be identified at acceptably low false discovery rates (FDRs). To meet estimated methyl-PSM FDRs, methyl-PSM filtering criteria are often determined using the target-decoy approach. The efficacy of this methyl-PSM filtering approach has, however, yet to be thoroughly evaluated. Here, we conduct a systematic analysis of methyl-PSM FDRs across a range of sample preparation workflows (each differing in their exposure to the alcohols methanol and isopropyl alcohol) and mass spectrometric instrument platforms (each employing a different mode of MS/MS dissociation). Through (13)CD3-methionine labeling (heavy-methyl SILAC) of Saccharomyces cerevisiae cells and in-depth manual data inspection, accurate lists of true positive methyl-PSMs were determined, allowing methyl-PSM FDRs to be compared with target-decoy approach-derived methyl-PSM FDR estimates. These results show that global FDR estimates produce extremely unreliable methyl-PSM filtering criteria; we demonstrate that this is an unavoidable consequence of the high number of amino acid combinations capable of producing peptide sequences that are isobaric to methylated peptides of a different sequence. Separate methyl-PSM FDR estimates were also found to be unreliable due to prevalent sources of false positive methyl-PSMs that produce high peptide identity score distributions. Incorrect methylation site localizations, peptides containing cysteinyl-S-β-propionamide, and methylated glutamic or aspartic acid residues can partially, but not wholly, account for these false positive methyl-PSMs. Together, these results indicate that the target-decoy approach is an unreliable means of estimating methyl-PSM FDRs and methyl-PSM filtering criteria. We suggest that orthogonal methylpeptide validation (e.g. heavy-methyl SILAC or its offshoots) should be considered a prerequisite for obtaining high confidence methyl-PSMs in large scale LC-MS/MS methylation site discovery experiments and make recommendations on how to reduce methyl-PSM FDRs in samples not amenable to heavy isotope labeling. Data are available via ProteomeXchange with the data identifier PXD002857.
authors.affiliation: From the ‡New South Wales Systems Biology Initiative, School of Biotechnology and Biomolecular Sciences, and g.hart-smith@unsw.edu.au.
authors.forename: Gene
authors.initials: G
authors.lastname: Hart-Smith
authors.affiliation: From the ‡New South Wales Systems Biology Initiative, School of Biotechnology and Biomolecular Sciences, and.
authors.forename: Daniel
authors.initials: D
authors.lastname: Yagoub
authors.affiliation: From the ‡New South Wales Systems Biology Initiative, School of Biotechnology and Biomolecular Sciences, and.
authors.forename: Aidan P
authors.initials: AP
authors.lastname: Tay
authors.affiliation: ‖Bioanalytical Mass Spectrometry Facility, University of New South Wales, Sydney, New South Wales 2052, Australia.
authors.forename: Russell
authors.initials: R
authors.lastname: Pickford
authors.affiliation: From the ‡New South Wales Systems Biology Initiative, School of Biotechnology and Biomolecular Sciences, and.
authors.forename: Marc R
authors.initials: MR
authors.lastname: Wilkins
mesh_terms: D005189:False Positive Reactions; D008745:Methylation; D010455:Peptides / Q000737:chemistry; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods
chemical_list: D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins