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 CD 3 -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 methylPSMs that produce high peptide identity score distributions. Incorrect methylation site localizations, peptides containing cysteinyl-S-␤-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- Post-translational methylation is a widespread protein modification, which predominantly occurs on lysine and arginine residues (1). Protein-lysine methyltransferases catalyze the methylation of lysine residues; these enzymes facilitate the incorporation of methyl groups into the N atoms of lysine residues to produce either mono-, di-, or tri-methyllysine (MML, 1 DML, and TML, respectively). Protein-arginine methyltransferases catalyze the methylation of arginine residues; these enzymes primarily act upon N G atoms to produce mono, asymmetric di-, or symmetric di-methylarginine, although the enzyme-mediated modification of N ␦ atoms to produce ␦-MMA has also been reported in Saccharomyces cerevisiae (2).Traditionally, lysine and arginine methylation have been closely associated with histone proteins, and their crucial roles in modifying chromatin structure have been extensively studied (3). In recent years, however, a growing number of large scale methylation site discovery experiments have indiFrom the ‡New South

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