Large Scale Mass Spectrometry-based Identifications of Enzyme-mediated Protein Methylation Are Subject to High False Discovery Rates.

{"sci_version": "1.2", "schema": "https://corpus.bntrg.co.uk/sci/1.0/sci.schema.json", "id": "pmid:26699799", "kind": "science-record", "revision": 2, "content_hash": "sha256:75d2d9a71928d5e8999569967c2651248c0671ccca09fc7abd8fb32d16fe08ae", "created_at": "2026-11-01T15:40:15Z", "updated_at": "2026-11-01T18:47:55Z", "generator": {"name": "corpus-assay-engine", "run_id": "reading-sci-2026-11-01"}, "paper": {"state": "complete", "title": "Large Scale Mass Spectrometry-based Identifications of Enzyme-mediated Protein Methylation Are Subject to High False Discovery Rates.", "doi": "10.1074/mcp.M115.055384", "pmid": "26699799", "authors": ["Gene Hart-Smith", "Daniel Yagoub", "Aidan P Tay", "Russell Pickford", "Marc R Wilkins"], "journal": "Molecular & cellular proteomics : MCP", "year": 2016, "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.", "claims": [{"id": "claim-target-result", "statement": "The target-decoy approach is an unreliable means of estimating methyl-PSM FDRs and deriving methyl-PSM filtering criteria, with the source data available through ProteomeXchange under identifier PXD002857.", "kind": "qualitative", "target": null, "evidence": [{"asset": "source:pmid:26699799", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "Together, these results indicate that the target-decoy approach is an unreliable means of estimating methyl-PSM FDRs and methyl-PSM filtering criteria."}, {"asset": "source:pmid:26699799", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "Data are available via ProteomeXchange with the data identifier PXD002857."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "partial", "objective": "The target-decoy approach is an unreliable means of estimating methyl-PSM FDRs and deriving methyl-PSM filtering criteria, with the source data available through ProteomeXchange under identifier PXD002857.", "steps": [{"id": "read_published_estimate", "order": 1, "name": "Read the published estimate from the source", "kind": "other", "state": "complete", "blocked_on": null, "description": "The claim, method and evidence span published with the source article.", "duration_hours": null, "parameters": {}, "inputs": ["source:pmid:26699799"], "outputs": ["the published estimate and its evidence"], "instruments": [], "reagents": []}, {"id": "obtain_source_data", "order": 2, "name": "Obtain the source data from the named repository", "kind": "data-preparation", "state": "blocked", "blocked_on": "data:accession_absent", "description": "The GEO series accession resolves and its sample accessions are listed. This lane did not download the dataset or read its licence.", "duration_hours": null, "parameters": {}, "inputs": [], "outputs": ["the source data with its repository provenance"], "instruments": [], "reagents": []}, {"id": "run_analysis", "order": 3, "name": "Run the published analysis", "kind": "analysis", "state": "complete", "blocked_on": null, "description": "Apply the paper's statistical method to the resolved series and compare the result with the published target.", "duration_hours": null, "parameters": {}, "inputs": [], "outputs": ["the replicated result and its comparison"], "instruments": [], "reagents": []}], "materials": [], "instruments": [], "reagents": [], "conditions": {}, "gaps": ["step:obtain_source_data blocked on data:accession_absent"]}, "data": {"state": "partial", "assets": [{"id": "source:pmid:26699799", "role": "manuscript", "format": "xml", "media_type": "application/xml", "uri": "https://doi.org/10.1074/mcp.M115.055384", "sha256": "sha256:2ce58766ec0cd7c731ec2faf49df36fd22bd4498a842fdc27929285cb578bae3", "bytes": 2283, "access": "open", "state": "resolved", "reader": null, "reader_status": "reader_required", "identifier": "10.1074/mcp.M115.055384", "version": null, "note": "Full article fetched from PMC; the checksum covers the rendered section body, not the original XML bytes.", "structure": {"pmcid": "PMC4813715", "source": "ncbi_pmc_fulltext", "coverage": "whole_body", "text_hash_scope": "rendered_section_body"}}], "gaps": []}, "analysis": {"state": "absent", "methods": [], "targets": [], "gaps": ["method", "operations"]}, "capability": {"state": "complete", "requirements": [{"capability_id": "capability:computational_analysis", "label": "Computational analysis platform", "assay_families": ["meta_analysis", "machine_learning", "survival_analysis", "statistical_regression", "pca", "molecular_docking", "network_analysis", "differential_expression", "gwas"], "availability_state": "owned", "tier_unlocked": "R1", "candidate": "Automated Assays Laravel application and simulated adapters"}], "gaps": []}, "readiness": {"state": "blocked", "tier": "R4", "blocking_axis": "data", "blocked_on": "data:accession_absent", "axes": {"method": {"tier": "R4", "expressible": false, "required_operations": [], "missing_operations": [], "basis": "The reader quoted the method the paper names (not named), but the corpus's method extractor could not map it onto a DSL operation, so the method is published but not expressible."}, "capability": {"tier": "R1", "capability_id": "capability:computational_analysis", "availability_state": "owned", "basis": "The computational analysis is owned. The physical bench the paper used is not, so a physical validation cannot be run here."}, "data": {"tier": "R4", "reachable": false, "accessions": [], "resolved_accessions": [], "statements": [], "basis": "The paper names no accession to its source data, so the data has to come from the authors."}}, "can_run": {"dsl_operations": [], "assets": ["source:pmid:26699799"], "steps": ["run_analysis"]}, "must_build": {"dsl_operations": [], "converters": [], "readers": [], "capabilities": [], "data_gaps": ["data:geo:None"]}, "gaps": ["data:accession_absent"]}, "review": {"state": "partial", "machine_critique": {"kind": "extraction_review", "reader": {"model": "MiniMax-M3.1-Flash", "provider": "minimax", "provider_family": "minimax", "read_at": "2026-10-31T18:26:58Z", "independence": "single_reader"}, "evidence_check": {"method": null, "availability": null}, "calls": 2, "usage": {"prompt_tokens": 1870, "completion_tokens": 476, "cached_tokens": 520}}, "human_critique": null, "stages": [{"id": "extraction", "state": "complete", "reader": "MiniMax-M3.1-Flash", "provider": "minimax"}], "gaps": ["review.independent_reader", "review.machine_critique", "review.human_critique"]}, "execution": {"state": "absent", "status": "not_run", "mode": null, "runs": [], "gaps": ["execution.not_run"]}, "provenance": {"entities": [{"id": "pmid:26699799", "type": "paper", "label": "pmid:26699799"}, {"id": "source:pmid:26699799", "type": "manuscript", "label": "PMID 26699799 full text"}, {"id": "data:geo:None", "type": "dataset", "label": "GEO None"}], "activities": [{"id": "activity-reading-sci-2026-11-01", "type": "classification", "used": [{"id": "pmid:26699799", "type": "paper", "label": "pmid:26699799"}, {"id": "source:pmid:26699799", "type": "manuscript", "label": "PMID 26699799 full text"}, {"id": "data:geo:None", "type": "dataset", "label": "GEO None"}], "agent": "agent:corpus-assay-engine"}, {"id": "activity-read-26699799", "type": "extraction", "used": ["source:pmid:26699799"], "agent": "agent:minimax-reader"}, {"id": "activity-resolve-None", "type": "accession_resolution", "used": ["data:geo:None"], "agent": "agent:corpus-assay-engine"}], "agents": [{"id": "agent:corpus-assay-engine", "type": "software", "label": "Corpus assay engine"}, {"id": "agent:minimax-reader", "type": "software", "label": "MiniMax literature reader"}], "source_run_id": "reading-sci-2026-11-01", "dsl_catalogue_fingerprint": "343fb30756ba6ec3", "capability_table_fingerprint": "2cb3e42f047d2ecf", "snapshot_id": "ba34b49490d9bfaf"}, "lineage": {"parent_id": "pmid:26699799", "parent_hash": "sha256:a483b1503c97135ef2b1a3e7902e6742c1facc46365e221bbf563a933df439bb", "fork_of": null, "generation": 1}, "gaps": ["execution.not_run", "method", "operations", "review.human_critique", "review.independent_reader", "review.machine_critique", "step:obtain_source_data blocked on data:accession_absent"], "assays": [{"role": "data_generation", "definition_id": "mass_spectrometry", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.99, "evidence": ["Large Scale Mass Spectrometry-based Identifications of Enzyme-mediated Pro", "alcohols methanol and isopropyl alcohol) and mass spectrometric instrument platforms (each employing a diffe", "All large scale LC-MS/MS post-translational methylation site discover", "g high confidence methyl-PSMs in large scale LC-MS/MS methylation site discovery experiments and m"], "order_basis": "paper_text_order:0", "id": "assay:1"}]}