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

• Published in: Angewandte Chemie International Edition Vol. 62; no. 34; pp. e202303415 - n/a
• Main Authors: Truong, Thy, Webber, Kei G. I., Madisyn Johnston, S., Boekweg, Hannah, Lindgren, Caleb M., Liang, Yiran, Nydegger, Alissia, Xie, Xiaofeng, Tsang, Tsz‐Ming, Jayatunge, D. A. Dasun N., Andersen, Joshua L., Payne, Samuel H., Kelly, Ryan T.
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 21.08.2023
• Edition: International ed. in English
• Subjects: Autophagy; Biodegradation; Chromatography, Liquid - methods; Data acquisition; HeLa Cells; Humans; Innate immunity; Labels; LFQ; Liquid chromatography; nanoLC; nanoPOTS; Precursors; Protein transport; Proteins; Proteome - analysis; Proteomes; Proteomics; Proteomics - methods; Sample preparation; single-cell proteomics; Standard data; autophagy; single-cell proteomics; nanoLC; LFQ; nanoPOTS
• ISSN: 1433-7851; 1521-3773; 1521-3773
• DOI: 10.1002/anie.202303415

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. A new data acquisition strategy for biological mass spectrometry, termed wide‐window acquisition, combined with efficient sample preparation and ultra‐low‐flow liquid chromatography, enables single‐cell proteome profiling to an unprecedented depth of >3000 proteins per cell. This advance allows additional classes of proteins to be studied within single cells.