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

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 & AP;40 % relative to standard data-dependent acquisition. For a 40-min LC gradient operated at & AP;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.

Automated summary

We developed a new method called wide window acquisition (WWA) that combines efficient sample preparation and ultra-low-flow liquid chromatography to quantify >3,000 proteins from single cells in rapid label-free analyses. WWA increased the number of identified proteins by about 40% compared to standard data-dependent acquisition. Utilizing this platform, we compared protein expression in HeLa cells with and without the essential autophagy gene atg9a, and found significant changes in 268 proteins related to innate immunity, vesicle trafficking, and protein degradation.