{"doi":"10.1016/j.mcpro.2024.100790","title":"Fully Automated Workflow for Integrated Sample Digestion and Evotip Loading Enabling High-Throughput Clinical Proteomics","abstract":null,"journal":"Molecular & Cellular Proteomics","year":2024,"id":640578,"datarank":0.6090664515819629,"base_score":4.060443010546419,"endowment":4.060443010546419,"self_citation_contribution":0.6090664515819629,"citation_network_contribution":0.0,"self_endowment_contribution":0.6090664515819629,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":57,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1664889,"name":"Florian Harking","orcid":null,"position":1,"is_corresponding":false},{"id":1664891,"name":"Joel Mario Vej-Nielsen","orcid":"0000-0001-6800-5092","position":2,"is_corresponding":false},{"id":1664893,"name":"Magnus Huusfeldt","orcid":null,"position":3,"is_corresponding":false},{"id":252474,"name":"Dorte B. 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To address this issue, we prepared and evaluated a simple and efficient workflow on the Opentrons OT-2 robot that combines sample digestion, cleanup, and loading on Evotips in a fully automated manner, allowing the processing of up to 192 samples in 6 h. Analysis of 192 automated HeLa cell sample preparations consistently identified ∼8000 protein groups and ∼130,000 peptide precursors with an 11.5 min active liquid chromatography gradient with the Evosep One and narrow-window data-independent acquisition (nDIA) with the Orbitrap Astral mass spectrometer providing a throughput of 100 samples per day. Our results demonstrate a highly sensitive workflow yielding both reproducibility and stability at low sample inputs. The workflow is optimized for minimal sample starting amount to reduce the costs for reagents needed for sample preparation, which is critical when analyzing large biological cohorts. Building on the digesting workflow, we incorporated an automated phosphopeptide enrichment step using magnetic titanium-immobilized metal ion affinity chromatography beads. This allows for a fully automated proteome and phosphoproteome sample preparation in a single step with high sensitivity. Using the integrated digestion and Evotip loading workflow, we evaluated the effects of cancer immune therapy on the plasma proteome in metastatic melanoma patients.","is_dataset_classified":null,"base_score":0.0,"endowment":0.0,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"38777088","pmcid":"PMC11251069","openalex_id":null,"authors":[],"funders":[{"funder_name":"Innovationsfonden","grant_id":"ERA-PerMed-JTC2022-OVA-PDM","title":null},{"funder_name":"European Research Council","grant_id":"HighResCells-810057","title":null},{"funder_name":"European Commission Marie Sklodowska-Curie Actions","grant_id":"PUSHH-861389","title":null},{"funder_name":"Novo Nordisk Foundation","grant_id":"NNF20SA0064340","title":null},{"funder_name":"Novo Nordisk Foundation","grant_id":"NNF14CC0001","title":null},{"funder_name":"H2020","grant_id":"EPIC-XS-823839","title":null},{"funder_name":"European Research Council","grant_id":"810057","title":"A synergistic approach toward understanding receptor signaling in the cell at very high resolution"},{"funder_name":"European Commission","grant_id":"861389","title":"Palaeoproteomics to Unleash Studies on Human History"},{"funder_name":"European Commission","grant_id":"823839","title":"European Proteomics Infrastructure Consortium providing Access"},{"funder_name":"Novo Nordisk Foundation","grant_id":"unidentified","title":"unidentified"},{"funder_name":"Horizon 2020 Framework Programme","grant_id":"","title":null},{"funder_name":"European Research Council","grant_id":"","title":null}],"total_grants":12,"fwci":null,"citation_percentile":null,"influential_citations":0,"citation_trend":[],"oa_status":"gold","license":"cc-by","oa_locations":[{"url":"https://www.mcponline.org/article/S1535-9476(24)00080-X/pdf","host_type":"publisher"},{"url":"https://api.elsevier.com/content/article/PII:S153594762400080X?httpAccept=text/xml","host_type":"publisher"},{"url":"https://api.elsevier.com/content/article/PII:S153594762400080X?httpAccept=text/plain","host_type":"publisher"},{"url":"https://pmc.ncbi.nlm.nih.gov/articles/PMC11251069/pdf/main.pdf","host_type":"repository"},{"url":"https://researchprofiles.ku.dk/da/publications/3d7b6045-b2ea-43e4-b1b9-f44ade9d4146","host_type":"repository"},{"url":"https://europepmc.org/articles/PMC11251069","host_type":"Europe_PMC"},{"url":"https://europepmc.org/articles/PMC11251069?pdf=render","host_type":"Europe_PMC"},{"url":"https://doi.org/10.1101/2023.12.22.573056","host_type":""},{"url":"https://doi.org/10.1016/j.mcpro.2024.100790","host_type":""},{"url":"https://pubmed.ncbi.nlm.nih.gov/38777088","host_type":""},{"url":"http://dx.doi.org/10.1016/j.mcpro.2024.100790","host_type":""},{"url":"https://curis.ku.dk/ws/files/397985292/1_s2.0_S153594762400080X_main.pdf","host_type":""},{"url":"https://doi.org/https://doi.org/10.1016/j.mcpro.2024.100790","host_type":""}],"fields_of_study":["0301 basic medicine","0303 health sciences","03 medical and health sciences"],"mesh_terms":["Hela Cells","Humans","Melanoma","Phosphopeptides","Proteome","Chromatography, Liquid","Reproducibility of Results","Proteomics","Automation","High-Throughput Screening Assays","Workflow"],"keywords":["Mass spectrometry","Plasma Proteomics","Biomarker Discovery","Workflow Automation","Cancer Immune Therapy","Proteomics","Phosphopeptides","Proteome","Reproducibility of Results","Workflow","High-Throughput Screening Assays","Automation","Humans","Research Article Collection: Advances in Proteomics Technology","Melanoma","HeLa Cells","Chromatography, Liquid"],"sdg_mappings":[{"sdg_number":3,"sdg_label":"3. 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