Insights from the First Phosphopeptide Challenge of the MS Resource Pillar of the HUPO Human Proteome Project.

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Despite considerable progress, the correct identification of phosphorylated sites, their quantification, and their interpretation regarding physiological relevance remain challenging. The MS Resource Pillar of the Human Proteome Organization (HUPO) Human Proteome Project (HPP) initiated the Phosphopeptide Challenge as a resource to help the community evaluate methods, learn procedures and data analysis routines, and establish their own workflows by comparing results obtained from a standard set of 94 phosphopeptides (serine, threonine, tyrosine) and their nonphosphorylated counterparts mixed at different ratios in a neat sample and a yeast background. Participants analyzed both samples with their method(s) of choice to report the identification and site localization of these peptides, determine their relative abundances, and enrich for the phosphorylated peptides in the yeast background. We discuss the results from 22 laboratories that used a range of different methods, instruments, and analysis software. We reanalyzed submitted data with a single software pipeline and highlight the successes and challenges in correct phosphosite localization. All of the data from this collaborative endeavor are shared as a resource to encourage the development of even better methods and tools for diverse phosphoproteomic applications. All submitted data and search results were uploaded to MassIVE (https://massive.ucsd.edu/) as data set MSV000085932 with ProteomeXchange identifier PXD020801.", "claims": [{"id": "claim-data-access", "statement": "All report submissions and their data are available on ProteomeXchange28 (PXD020801), and, where possible, the data files can be directly examined in MassIVE.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:33166149", "locator": "sections[16] \"CONCLUSIONS\" paragraph 2", "quote": "All report submissions and their data are available on ProteomeXchange28 (PXD020801), and, where possible, the data files can be directly examined in MassIVE."}], "state": "complete"}, {"id": "claim-target-result", "statement": "Across most reports, phosphopeptide identification and site-localization performance was relatively high, but precision and sensitivity varied substantially and localization was especially difficult for multiply phosphorylated peptides.", "kind": "qualitative", "target": null, "evidence": [{"asset": "source:pmid:33166149", "locator": "sections[11] \"Challenge #1\" paragraph 5", "quote": "However, it is worth noting that within this cluster, the ranges of precision (73–99%) and sensitivity (69–94%) are somewhat wide."}, {"asset": "source:pmid:33166149", "locator": "sections[11] \"Challenge #1\" paragraph 6", "quote": "Overall, correct localization is clearly more difficult in peptides with several possible phosphorylation sites, especially when these are adjacent or in close proximity, that is, less than three amino acids apart."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "partial", "objective": "Across most reports, phosphopeptide identification and site-localization performance was relatively high, but precision and sensitivity varied substantially and localization was especially difficult for multiply phosphorylated peptides.", "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:33166149"], "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_unresolved", "description": "The GEO series accession resolves and its sample accessions are listed. 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