biostudies-literatureJiang YNational Institute of General Medical Sciences677-685https://www.ebi.ac.uk/biostudies/studies/S-EPMC9850400biostudies-literatureUnknown95(2)Large-scale proteome analysis requires rapid and high-throughput analytical methods. We recently reported a new paradigm in proteome analysis where direct infusion and ion mobility are used instead of liquid chromatography (LC) to achieve rapid and high-throughput proteome analysis. Here, we introduce an improved direct infusion shotgun proteome analysis protocol including label-free quantification (DISPA-LFQ) using CsoDIAq software. With CsoDIAq analysis of DISPA data, we can now identify up to ∼2000 proteins from the HeLa and 293T proteomes, and with DISPA-LFQ, we can quantify ∼1000 proteins from no more than 1 μg of sample within minutes. The identified proteins are involved in numerous valuable pathways including central carbon metabolism, nucleic acid replication and transport, protein synthesis, and endocytosis. Together with a high-throughput sample preparation method in a 96-well plate, we further demonstrate the utility of this technology for performing high-throughput drug analysis in human 293T cells. The total time for data collection from a whole 96-well plate is approximately 8 h. We conclude that the DISPA-LFQ strategy presents a valuable tool for fast identification and quantification of proteins in complex mixtures, which will power a high-throughput proteomic era of drug screening, biomarker discovery, and clinical analysis.Analytical chemistryLabel-Free Quantification from Direct Infusion Shotgun Proteome Analysis (DISPA-LFQ) with CsoDIAq Software.PMC9850400R35GM142502Jiang YMeyer JGCranney CWHutton AfalseLabel-Free Quantification from Direct Infusion Shotgun Proteome Analysis (DISPA-LFQ) with CsoDIAq Software.Large-scale proteome analysis requires rapid and high-throughput analytical methods. We recently reported a new paradigm in proteome analysis where direct infusion and ion mobility are used instead of liquid chromatography (LC) to achieve rapid and high-throughput proteome analysis. Here, we introduce an improved direct infusion shotgun proteome analysis protocol including label-free quantification (DISPA-LFQ) using CsoDIAq software. With CsoDIAq analysis of DISPA data, we can now identify up to ∼2000 proteins from the HeLa and 293T proteomes, and with DISPA-LFQ, we can quantify ∼1000 proteins from no more than 1 μg of sample within minutes. The identified proteins are involved in numerous valuable pathways including central carbon metabolism, nucleic acid replication and transport, protein synthesis, and endocytosis. Together with a high-throughput sample preparation method in a 96-well plate, we further demonstrate the utility of this technology for performing high-throughput drug analysis in human 293T cells. The total time for data collection from a whole 96-well plate is approximately 8 h. We conclude that the DISPA-LFQ strategy presents a valuable tool for fast identification and quantification of proteins in complex mixtures, which will power a high-throughput proteomic era of drug screening, biomarker discovery, and clinical analysis.2023-01-01T00:00:00Z2023 Jan2024-11-09T02:23:33.47Z2024-11-09T02:23:33.47ZS-EPMC98504003652771810.1021/acs.analchem.2c02249