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Rapid, One-Step Sample Processing for Label-Free Single-Cell Proteomics
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DOI：10.1021/jasms.3c00159.png)
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Sample preparationfor single-cell proteomics is generally performedin a one-pot workflow with multiple dispensing and incubation steps.These hours-long processes can be labor intensive and lead to longsample-to-answer times. Here we report a sample preparation methodthat achieves cell lysis, protein denaturation, and digestion in 1h using commercially available high-temperature-stabilized proteaseswith a single reagent dispensing step. Four different one-step reagentcompositions were evaluated, and the mixture providing the highestproteome coverage was compared to the previously employed multistepworkflow. The one-step preparation increases proteome coverage relativeto the previous multistep workflow while minimizing labor input andthe possibility of human error. We also compared sample recovery betweenpreviously used microfabricated glass nanowell chips and injection-moldedpolypropylene chips and found the polypropylene provided improvedproteome coverage. Combined, the one-step sample preparation and thepolypropylene substrates enabled the identification of an averageof nearly 2400 proteins per cell using a standard data-dependent workflowwith Orbitrap mass spectrometers. These advances greatly simplifysample preparation for single-cell proteomics and broaden accessibilitywith no compromise in terms of proteome coverage.
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MASS-SPECTROMETRY
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DISCOVERY
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2.7
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