Mining Missing Membrane Proteins by High-pH Reverse-Phase StageTip Fractionation and Multiple Reaction Monitoring Mass Spectrometry.

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Over one-third of these missing proteins are annotated as membrane proteins, owing to their relatively challenging accessibility with standard shotgun proteomics. Using nonsmall cell lung cancer (NSCLC) as a model study, we aim to mine missing proteins from disease-associated membrane proteome, which may be still largely under-represented. To increase identification coverage, we employed Hp-RP StageTip prefractionation of membrane-enriched samples from 11 NSCLC cell lines. Analysis of membrane samples from 20 pairs of tumor and adjacent normal lung tissue was incorporated to include physiologically expressed membrane proteins. Using multiple search engines (X!Tandem, Comet, and Mascot) and stringent evaluation of FDR (MAYU and PeptideShaker), we identified 7702 proteins (66% membrane proteins) and 178 missing proteins (74 membrane proteins) with PSM-, peptide-, and protein-level FDR of 1%. Through multiple reaction monitoring using synthetic peptides, we provided additional evidence of eight missing proteins including seven with transmembrane helix domains. This study demonstrates that mining missing proteins focused on cancer membrane subproteome can greatly contribute to map the whole human proteome. All data were deposited into ProteomeXchange with the identifier PXD002224.", "claims": [{"id": "claim-target-result", "statement": "Using multiple search engines and stringent false discovery rate evaluation, the study identified 7,702 proteins, including 178 missing proteins, and deposited the data in ProteomeXchange under identifier PXD002224.", "kind": "qualitative", "target": null, "evidence": [{"asset": "source:pmid:26202522", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "Using multiple search engines (X!Tandem, Comet and Mascot) and stringent evaluation of FDR (MAYU and PeptideShaker), we identified 7702 proteins (66% membrane proteins) and 178 missing proteins (74 membrane proteins) with PSM-, peptide-, and protein-level FDR of 1%."}, {"asset": "source:pmid:26202522", "locator": "sections[0] \"Abstract\" paragraph 1", "quote": "All data were deposited into ProteomeXchange with the identifier PXD002224."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "partial", "objective": "Using multiple search engines and stringent false discovery rate evaluation, the study identified 7,702 proteins, including 178 missing proteins, and deposited the data in ProteomeXchange under identifier PXD002224.", "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:26202522"], "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. 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In order to check the possibility", "llenging accessibility with standard shotgun proteomics. Using nonsmall cell lung cancer (NSCLC) as"], "order_basis": "paper_text_order:69", "id": "assay:1"}, {"role": "data_generation", "definition_id": "cell_culture", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.99, "evidence": ["on The human primary lung cancer cell lines: A549, CL100, CL141, CL152, CL25, CL83, CL97, H197", "change (SAX) StageTip was demonstrated using HeLa cell lines.24 Comparison with the Peptide At"], "order_basis": "paper_text_order:2855", "id": "assay:2"}]}