Post-translational methylation is a protein modification that predominantly occurs on lysine and arginine residues, and a growing number of recent large-scale LC-MS/MS methylation site discovery experiments have indicated that these modifications are widespread and sometimes conserved in eukaryotes. In interpreting these LC-MS/MS data, methylpeptide spectrum matches (methyl-PSMs) must be identified at acceptably low false discovery rates (FDRs) following sequence database searching, and to estimate FDRs and determine methyl-PSM filtering criteria, the target-decoy approach is frequently employed. 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 and mass spectrometric instrument platforms (each employing a different mode of MS/MS dissociation). Through ¹³CD₃-methionine labeling (heavy-methyl SILAC) of S. cerevisiae cells and in-depth manual data inspection, accurate lists of true positive methyl-PSMs were determined, allowing methyl-PSM FDRs to be compared to target-decoy approach-derived methyl-PSM FDR estimates.

Our results show that global FDR estimates (i.e. FDR estimates obtained using all PSMs from target and decoy databases) 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 (i.e. FDR estimates obtained using only methyl-PSMs from target and decoy databases) 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) should be considered a prerequisite for obtaining high confidence methyl-PSMs in large-scale LC-MS/MS methylation site discovery experiments.