Optimization of Ultrafast Proteomics Using an LC-Quadrupole-Orbitrap Mass Spectrometer with Data-Independent Acquisition

Ishikawa, M.; Konno, Ryuichi; Nakajima, D.; Gotoh, Mari; Fukasawa, Keiko; Sato, Hironori; Nakamura, Ren; Ohara, Osamu; Kawashima, Yusuke

doi:10.1021/acs.jproteome.2c00121

Cited by 26 publications

(25 citation statements)

References 31 publications

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“…Conversely, with a 5-fold increase in selectivity at the same number of cysteines profiled, 50% of druggable cysteines would be profiled. Consistent with recent advances in high-throughput proteome profiling and instrumental advances, [40][41][42][43] peptide detection improvements in the coming years will no doubt improve the number of cysteines profiled in a given MS-ABPP experiment. Combined with technical advances in sample preparation, acquisition and analysis to alleviate known limitations of bottom-up approaches (e.g.…”

Section: Implications For Cysteine Profiling In Targeted Covalent Inh...mentioning

confidence: 78%

Proteome-wide structure-based accessibility analysis of ligandable and detectable cysteines in chemoproteomic datasets

White

Gil

Tate

2022

Preprint

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Covalent drug discovery, in particular targeting reactive cysteines, has undergone a resurgence over the past two decades, demonstrated by recent clinical successes of covalent inhibitors for high-priority cancer targets. Reactive cysteine profiling, first pioneered by the Cravatt lab, has emerged in parallel as a powerful approach for proteome-wide on- and off-target profiling. Thus far however, structural analysis of liganded cysteines has been restricted to experimentally determined protein structures. We combined AlphaFold-predicted amino acid side chain accessibilities for >95% of the human proteome with a meta-analysis of thirteen public cysteine profiling datasets, totalling 40,070 unique cysteine residues, revealing accessibility biases in sampled cysteines primarily dictated by warhead chemistry. Analysis of >3.5 million cysteine-fragment interactions further suggests that exposed cysteine residues are preferentially targeted by elaborated fragments and drug-like compounds. We finally propose a framework for benchmarking coverage of ligandable cysteines in future cysteine profiling approaches, considering both selectivity for high-priority residues and quantitative depth. All analysis and produced resources (freely available at www.github.com/TateLab) are readily extendable to reactive amino acids beyond cysteine, and related questions in chemical biology.

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Section: Implications For Cysteine Profiling In Targeted Covalent Inh...mentioning

confidence: 78%

Proteome-wide structure-based accessibility analysis of ligandable and detectable cysteines in chemoproteomic datasets

White

Gil

Tate

2022

Preprint

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“…There are multiple competing fast proteomics methods using LC–MS that can achieve more than 2000 protein identifications with minutes. ,,− However, time spent on sample loading, column equilibration, and column wash may not be included in reported times. If all time is counted, usually the maximum throughput for 24 h is less than 300 samples.…”

Section: Discussionmentioning

confidence: 99%

Label-Free Quantification from Direct Infusion Shotgun Proteome Analysis (DISPA-LFQ) with CsoDIAq Software

et al. 2022

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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.

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“…Peptide separation operated at flow rates of 1-10 mL min À1 might be a compromise approach. [153][154][155]…”

Section: Fast Peptide Separation For High-throughput Proteomic Analysismentioning

confidence: 99%

Chromatographic separation of peptides and proteins for characterization of proteomes

Liang

Zhang

2023

Chem. Commun.

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Optimization of Ultrafast Proteomics Using an LC-Quadrupole-Orbitrap Mass Spectrometer with Data-Independent Acquisition

Cited by 26 publications

References 31 publications

Proteome-wide structure-based accessibility analysis of ligandable and detectable cysteines in chemoproteomic datasets

Proteome-wide structure-based accessibility analysis of ligandable and detectable cysteines in chemoproteomic datasets

Label-Free Quantification from Direct Infusion Shotgun Proteome Analysis (DISPA-LFQ) with CsoDIAq Software

Chromatographic separation of peptides and proteins for characterization of proteomes

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