Dataset Information

P-VIS: internally-controlled validation of mass spectrometry-based peptide identifications

ABSTRACT: Peptide-Spectrum Match (PSM) Validation with Internal Standards (P-VIS) is an objective approach to validating individual PSMs. A biological sample is analyzed by LC-MS/MS and the data are searched using a traditional approach such as a database search or de novo sequencing. A PSM of interest is identified, and a synthetic version of the putative peptide is obtained. Internal standard peptides (ISPs) are spiked into both the biological sample and the validation peptide sample and both samples are analyzed by LC-MS/MS. Using PSM_validator (https://github.com/Delong-Lab/PSM_validator/releases), an open-source program we created to implement the P-VIS workflow, both the fragmentation spectrum and the chromatographic retention time for the peptide of interest in the biological sample are compared to those of the validation peptide. The same comparisons are made for each of the ISPs, and these results are used to establish a prediction interval for valid matches. If the values for the comparisons between the biological and validation peptide fall within the prediction intervals, the match is considered valid. This submission contains the data and P-VIS results from a study aimed at benchmarking the P-VIS workflow.

INSTRUMENT(S): 6550 iFunnel Q-TOF LC/MS

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

SUBMITTER: Timothy Wiles

LAB HEAD: Thomas Delong

PROVIDER: PXD020901 | Pride | 2020-09-21

REPOSITORIES: Pride

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Publications

Peptide-Spectrum Match Validation with Internal Standards (P-VIS): Internally-Controlled Validation of Mass Spectrometry-Based Peptide Identifications.

Wiles Timothy Aaron TA Saba Laura M LM Delong Thomas T

Journal of proteome research 20200929 1

Liquid chromatography-tandem mass spectrometry is an increasingly powerful tool for studying proteins in the context of disease. As technological advances in instrumentation and data analysis have enabled deeper profiling of proteomes and peptidomes, the need for a rigorous, standardized approach to validate individual peptide-spectrum matches (PSMs) has emerged. To address this need, we developed a novel and broadly applicable workflow: PSM validation with internal standards (P-VIS). In this ap ...[more]

PMID: 32924495

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Project 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.

Dataset Information

P-VIS: internally-controlled validation of mass spectrometry-based peptide identifications

Publications

Peptide-Spectrum Match Validation with Internal Standards (P-VIS): Internally-Controlled Validation of Mass Spectrometry-Based Peptide Identifications.

Similar Datasets

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