{"doi":"10.64898/2025.12.26.696642","title":"Improved surfactant-assisted one-pot sample preparation for robust convenient single cell proteomics","abstract":"Abstract Recent advances in mass spectrometry-based single-cell proteomics (SCP) technologies have revolutionized the SCP field for comprehensive characterization of cellular heterogeneity. However, most of the current SCP approaches employ sub-µL to 1 µL processing volume for effective single-cell sample preparation using either ultralow-volume specialized devices or a 384-well plate by frequently adding water to the plate well to compensate water evaporation, which greatly limits their broad accessibility and analytical robustness. Here we report a robust convenient SCP method termed iSOP ( i mproved S urfactant-assisted O ne- P ot processing) for processing of single cells at the low µL processing volume using the 384-well plate with tight sealing to avoid sample drying loss. This iSOP SCP method was built upon our previously developed SOP method by the replacement of a PCR tube or 96-well plate with the low-volume 384-well plate and systematic optimization of the single-cell processing conditions. After optimization, 3 µL was selected as the processing volume with a mixture of 2 ng trypsin and 2 ng Lys-C enzymes in terms of robustness, detection sensitivity, and operation convenience. With a commonly accessible LC-MS platform, iSOP-MS can detect and quantify ∼1,200–1,800 protein groups from single HeLa or MCF7 cells. Application of iSOP-MS to two neuroblastoma cell lines has demonstrated that iSOP-MS enabled reliable identification of an average of ∼1,700 and ∼2,050 protein groups from single BE2-C and SK-N-SH cells, respectively, and precise characterization of cellular heterogeneity between the two distinct cell types and within the same cell type. When compared to other available SCP methods, iSOP-MS is more robust and convenient for routine, cost-effective quantitative SCP analysis.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":587253,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9527,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"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":869702,"name":"Zhangyang Xu","orcid":"0000-0002-0314-9244","position":1,"is_corresponding":false},{"id":1405804,"name":"Nadia Bayou","orcid":null,"position":2,"is_corresponding":false},{"id":232133,"name":"Rui Zhao","orcid":"0000-0002-6602-6583","position":3,"is_corresponding":false},{"id":1502975,"name":"William B Christler","orcid":null,"position":4,"is_corresponding":false},{"id":326357,"name":"Matthew Gaffrey","orcid":"0009-0001-2436-6969","position":5,"is_corresponding":false},{"id":232117,"name":"Karl Weitz","orcid":"0000-0003-3453-1283","position":6,"is_corresponding":false},{"id":872481,"name":"Mara Serena Serafini","orcid":"0000-0001-9192-9170","position":7,"is_corresponding":false},{"id":1409997,"name":"Elisabetta Molteni","orcid":"0009-0005-1979-5020","position":8,"is_corresponding":false},{"id":60409,"name":"Vladislav Petyuk","orcid":"0000-0003-4076-151X","position":9,"is_corresponding":false},{"id":311674,"name":"Massimo Cristofanilli","orcid":"0000-0002-4194-7175","position":10,"is_corresponding":false},{"id":118835,"name":"T. LIU","orcid":null,"position":11,"is_corresponding":false},{"id":620530,"name":"Carolina Reduzzi","orcid":"0000-0003-0514-0152","position":12,"is_corresponding":false},{"id":291295,"name":"Tujin Shi","orcid":"0000-0002-5592-3588","position":13,"is_corresponding":false},{"id":869704,"name":"Reta Birhanu Kitata","orcid":"0000-0002-8973-7679","position":0,"is_corresponding":true}],"reference_count":41,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:59:36.020030Z","pmid":"41497649","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":[]}