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bioRxiv · 10.1101/2021.01.30.428333

Robust, sensitive, and quantitative single-cell proteomics based on ion mobility filtering

Woo, J.·Clair, G. C.·Williams, S. M.·Feng, S.·Moore, R. J.·Chrisler, W. B.·Smith, R. D.·Kelly, R. T.·Pasa-Tolic, L.·Ansong, C.·Zhu, Y.
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Abstract

Unbiased single-cell proteomics (scProteomics) promises to advance our understanding of cell functions within complex biological systems. However, a major challenge for current methods is their ability to identify and provide accurate quantitative information for low abundance proteins. Herein, we describe an ion mobility-enhanced mass spectrometry acquisition and peptide identification method, TIFF (Transferring Identification based on FAIMS Filtering), designed to improve the sensitivity and accuracy of label-free scProteomics. TIFF significantly extends the ion accumulation times for peptide ions by filtering out singly charged background ions. The peptide identities are then assigned by a 3-dimensional MS1 feature matching approach (retention time, accurate mass, and FAIMS compensation voltage). The TIFF method enabled unbiased proteome analysis to a depth of >1,700 proteins in single HeLa cells with >1,100 proteins consistently quantified. As a demonstration, we applied the TIFF method to obtain temporal proteome profiles of >150 single murine macrophage cells during a lipopolysaccharide stimulation experiment and identified time-dependent proteome profiles.

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Woo, J., Clair, G. C., Williams, S. M., Feng, S., Moore, R. J., Chrisler, W. B., Smith, R. D., Kelly, R. T., Pasa-Tolic, L., Ansong, C., Zhu, Y.. 2021-02-01. Robust, sensitive, and quantitative single-cell proteomics based on ion mobility filtering. https://doi.org/10.1101/2021.01.30.428333

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