{"doi":"10.1021/acs.jproteome.4c00892","title":"High-Throughput Workflow for Detergent-free Cell-Based Proteomic Characterization","abstract":"We have developed an automated cell-based workflow for the quantification of proteins by liquid chromatography-mass spectrometry (LC-MS) that facilitates large-scale perturbation studies carried out in a 96-well plate format and enables the preparation of one full plate in approximately 4 h, showcasing a high-throughput (HTP) concept. Cells were grown in a 96-well plate and lysed via ultrasonication. Proteins were subsequently solubilized, extracted, and processed into tryptic peptides for 2 h before being acquired by data-independent acquisition mass spectrometry (DIA-MS). This workflow leverages adaptive focused acoustics (AFA) technology for ultrasonication to aid cell lysis and protein solubilization on an automated liquid handling platform. As proof of principle, AC16 human cardiomyocyte-like cells were cultured in a 96-well plate under optimized conditions that were compatible with the downstream HTP pipeline. Over 30,000 peptides were identified, corresponding to the detection of 5100 unique proteins. 50% of measured proteins had an average coefficient of variation (CV) under 25% from approximately 30,000 cells. Our optimized detergent-free buffer consisting of ammonium bicarbonate yielded comparable findings. For the same number of cells, 5000 proteins were identified from 29,000 peptides, 40% of which demonstrated a CV under 25%.","journal":"Journal of Proteome Research","year":2025,"id":536116,"datarank":0.10397207708399181,"base_score":0.6931471805599453,"endowment":0.6931471805599453,"self_citation_contribution":0.10397207708399181,"citation_network_contribution":0.0,"self_endowment_contribution":0.10397207708399181,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9578,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":872353,"name":"Alejandro Rivas","orcid":"0000-0002-9613-3769","position":1,"is_corresponding":false},{"id":1219064,"name":"Maxim Zhgamadze","orcid":null,"position":2,"is_corresponding":false},{"id":873162,"name":"Ali Haghani","orcid":null,"position":3,"is_corresponding":false},{"id":326248,"name":"Simion Kreimer","orcid":"0000-0001-6627-3771","position":4,"is_corresponding":false},{"id":1420966,"name":"Ajay Bharadwaj","orcid":null,"position":5,"is_corresponding":false},{"id":471722,"name":"Niveda Sundararaman","orcid":"0000-0001-7708-3754","position":6,"is_corresponding":false},{"id":1420967,"name":"Sameer Vasantgadkar","orcid":null,"position":7,"is_corresponding":false},{"id":320687,"name":"Kasturi Pal","orcid":"0000-0002-7328-5153","position":8,"is_corresponding":false},{"id":582317,"name":"Eugenio Daviso","orcid":"0009-0005-8596-315X","position":9,"is_corresponding":false},{"id":372387,"name":"Aleksandr Stotland","orcid":"0000-0003-4794-1097","position":10,"is_corresponding":false},{"id":1420296,"name":"Christopher W. Murray","orcid":"0000-0003-3733-0700","position":11,"is_corresponding":false},{"id":584732,"name":"Koen Raedschelders","orcid":"0000-0003-0835-4762","position":12,"is_corresponding":false},{"id":1219065,"name":"Sudha S. Savant","orcid":null,"position":13,"is_corresponding":false},{"id":112291,"name":"Jennifer E. Van Eyk","orcid":"0000-0001-9050-148X","position":14,"is_corresponding":false},{"id":872352,"name":"Saeed Seyedmohammad","orcid":"0000-0001-8591-540X","position":0,"is_corresponding":true}],"reference_count":56,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:52:00.885532Z","pmid":"40255039","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}