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bioRxiv · 10.1101/2021.02.17.431689

High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip

Woo, J.·Williams, S. M.·Aguilera-Vazquez, V.·Sontag, R. L.·Moore, R. J.·Markillie, L. M.·Mehta, H. S.·Cantlon, J.·Adkins, J. N.·Smith, R. D.·Clair, G. C.·Pasa-Tolic, L.·Zhu, Y.
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Abstract

Global quantification of protein abundances in single cells would provide more direct information on cellular function phenotypes and complement transcriptomics measurements. However, single-cell proteomics (scProteomics) is still immature and confronts technical challenges, including limited proteome coverage, poor reproducibility, as well as low throughput. Here we describe a nested nanoPOTS (N2) chip to dramatically improve protein recovery, operation robustness, and processing throughput for isobaric-labeling-based scProteomics workflow. The N2 chip allows reducing cell digestion volume to <30 nL and increasing processing capacity to > 240 single cells in one microchip. In the analysis of [~]100 individual cells from three different cell lines, we demonstrate the N2 chip-based scProteomics platform can robustly quantify [~]1500 proteins and reveal functional differences. Our analysis also reveals low protein abundance variations (median CVs < 16.3%), highlighting the utility of such measurements, and also suggesting the single-cell proteome is highly stable for the cells cultured under identical conditions.

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Woo, J., Williams, S. M., Aguilera-Vazquez, V., Sontag, R. L., Moore, R. J., Markillie, L. M., Mehta, H. S., Cantlon, J., Adkins, J. N., Smith, R. D., Clair, G. C., Pasa-Tolic, L., Zhu, Y.. 2021-02-18. High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip. https://doi.org/10.1101/2021.02.17.431689

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