PXD002857

PXD002857 is an original dataset announced via ProteomeXchange.

Title	A systematic analysis of methylpeptide false discovery rates
Description	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 isopropanol) 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. 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.
HostingRepository	PRIDE
AnnounceDate	2016-01-05
AnnouncementXML	Submission_2016-01-05_13:11:56.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Gene Hart-Smith
SpeciesList	scientific name: Saccharomyces cerevisiae (Baker's yeast); NCBI TaxID: 4932;
ModificationList	monohydroxylated residue; iodoacetamide derivatized residue; trimethylated residue; monomethylated residue; dimethylated residue
Instrument	LTQ Orbitrap Velos; Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2015-09-07 01:24:04	ID requested
⏵ 1	2016-01-05 13:11:58	announced

Hart-Smith G, Yagoub D, Tay AP, Pickford R, Wilkins MR, Large Scale Mass Spectrometry-based Identifications of Enzyme-mediated Protein Methylation Are Subject to High False Discovery Rates. Mol Cell Proteomics, 15(3):989-1006(2016) [pubmed]

curator keyword: Technical

submitter keyword: arginine methylation, lysine methylation, target-decoy approach, heavy-methyl SILAC, false discovery rates

Marc Ronald Wilkins
contact affiliation	School of Biotechnology and Biomolecular Sciences, Systems Biology Initiative, UNSW, Australia
contact email	m.wilkins@unsw.edu.au
lab head
Gene Hart-Smith
contact affiliation	UNSW
contact email	g.hart-smith@unsw.edu.au
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD002857
     1. Label: PRIDE project
     2. Name: A systematic analysis of methylpeptide false discovery rates