Data-Dependent Acquisition with Precursor Coisolation Improves Proteome Coverage and Measurement Throughput for Label-Free Single-Cell Proteomics.

Autor: Truong T; Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, 84602, USA., Webber KGI; Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, 84602, USA., Madisyn Johnston S; Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, 84602, USA., Boekweg H; Department of Biology, Brigham Young University, Provo, UT, 84602, USA., Lindgren CM; Department of Biology, Brigham Young University, Provo, UT, 84602, USA., Liang Y; Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, 84602, USA., Nydegger A; Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, 84602, USA., Xie X; Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, 84602, USA., Tsang TM; Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, 84602, USA., Jayatunge DADN; Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, 84602, USA., Andersen JL; Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, 84602, USA., Payne SH; Department of Biology, Brigham Young University, Provo, UT, 84602, USA., Kelly RT; Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, 84602, USA.
Jazyk: angličtina
Zdroj: Angewandte Chemie (International ed. in English) [Angew Chem Int Ed Engl] 2023 Aug 21; Vol. 62 (34), pp. e202303415. Date of Electronic Publication: 2023 Jul 13.
DOI: 10.1002/anie.202303415
Abstrakt: We combined efficient sample preparation and ultra-low-flow liquid chromatography with a newly developed data acquisition and analysis scheme termed wide window acquisition (WWA) to quantify >3,000 proteins from single cells in rapid label-free analyses. WWA employs large isolation windows to intentionally co-isolate and co-fragment adjacent precursors along with the selected precursor. Optimized WWA increased the number of MS2-identified proteins by ≈40 % relative to standard data-dependent acquisition. For a 40-min LC gradient operated at ≈15 nL/min, we identified an average of 3,524 proteins per single-cell-sized aliquot of protein digest. Reducing the active gradient to 20 min resulted in a modest 10 % decrease in proteome coverage. Using this platform, we compared protein expression between single HeLa cells having an essential autophagy gene, atg9a, knocked out, with their isogenic WT parental line. Similar proteome coverage was observed, and 268 proteins were significantly up- or downregulated. Protein upregulation primarily related to innate immunity, vesicle trafficking and protein degradation.
(© 2023 Wiley-VCH Verlag GmbH.)
Databáze: MEDLINE
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