{"doi":"10.1101/2021.02.17.431689","title":"High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip","abstract":"Abstract Global quantification of protein abundances in single cells would provide more direct information on cellular function phenotypes and complement transcriptomics measurements. However, single-cell proteomics (scProteomics) is still immature and confronts technical challenges, including limited proteome coverage, poor reproducibility, as well as low throughput. Here we describe a nested nanoPOTS (N2) chip to dramatically improve protein recovery, operation robustness, and processing throughput for isobaric-labeling-based scProteomics workflow. The N2 chip allows reducing cell digestion volume to <30 nL and increasing processing capacity to > 240 single cells in one microchip. In the analysis of ∼100 individual cells from three different cell lines, we demonstrate the N2 chip-based scProteomics platform can robustly quantify ∼1500 proteins and reveal functional differences. Our analysis also reveals low protein abundance variations (median CVs < 16.3%), highlighting the utility of such measurements, and also suggesting the single-cell proteome is highly stable for the cells cultured under identical conditions.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2021,"id":213250,"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":28,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9558,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":623752,"name":"Sarah Williams","orcid":"0000-0001-5659-6470","position":1,"is_corresponding":false},{"id":623754,"name":"Victor Aguilera-Vazquez","orcid":"0009-0000-1927-6574","position":2,"is_corresponding":false},{"id":623755,"name":"Ryan Sontag","orcid":"0000-0003-0537-1495","position":3,"is_corresponding":false},{"id":60416,"name":"Ronald Moore","orcid":"0000-0003-2806-2855","position":4,"is_corresponding":false},{"id":623753,"name":"Lye Meng Markillie","orcid":"0000-0001-8275-4882","position":5,"is_corresponding":false},{"id":624746,"name":"Hardeep Mehta","orcid":null,"position":6,"is_corresponding":false},{"id":632921,"name":"Joshua Cantlon","orcid":"0000-0003-1700-3737","position":7,"is_corresponding":false},{"id":235317,"name":"Joshua Adkins","orcid":"0000-0003-0399-0700","position":8,"is_corresponding":false},{"id":45123,"name":"Richard Smith","orcid":"0000-0002-2381-2349","position":9,"is_corresponding":false},{"id":352735,"name":"Gérémy Clair","orcid":"0000-0003-0478-8520","position":10,"is_corresponding":false},{"id":257356,"name":"Ljiljana Paša‐Tolić","orcid":null,"position":11,"is_corresponding":false},{"id":4911,"name":"Ying Zhu","orcid":"0000-0002-5416-0566","position":12,"is_corresponding":false},{"id":623751,"name":"Jongmin Jacob Woo","orcid":"0000-0001-5569-9304","position":0,"is_corresponding":true}],"reference_count":36,"raw_metadata":null,"created_at":"2026-07-18T23:52:36.886828Z","pmid":null,"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":[]}