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DOE OSTI · 1828225

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

Woo, Jongmin (ORCID:0000000155699304)·Williams, Sarah M.·Markillie, Lye Meng (ORCID:0000000182754882)·Feng, Song (ORCID:0000000339839009)·Tsai, Chia-Feng·Aguilera-Vazquez, Victor·Sontag, Ryan L.·Moore, Ronald J. (ORCID:0000000328062855)·Hu, Dehong (ORCID:0000000239742963)·Mehta, Hardeep S.·Cantlon-Bruce, Joshua·Liu, Tao (ORCID:0000000195296550)·Adkins, Joshua N. (ORCID:0000000303990700)·Smith, Richard D. (ORCID:0000000223812349)·Clair, Geremy C. (ORCID:0000000304788520)·Pasa-Tolic, Ljiljana (ORCID:0000000198535457)·Zhu, Ying (ORCID:0000000254160566)
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Abstract

Abstract Global quantification of protein abundances in single cells could provide direct information on cellular phenotypes and complement transcriptomics measurements. However, single-cell proteomics is still immature and confronts many technical challenges. Herein we describe a nested nanoPOTS (N2) chip to improve protein recovery, operation robustness, and processing throughput for isobaric-labeling-based scProteomics workflow. The N2 chip reduces reaction volume to <30 nL and increases capacity to >240 single cells on a single microchip. The tandem mass tag (TMT) pooling step is simplified by adding a microliter droplet on the nested nanowells to combine labeled single-cell samples. In the analysis of ~100 individual cells from three different cell lines, we demonstrate that the N2 chip-based scProteomics platform can robustly quantify ~1500 proteins and reveal membrane protein markers. Our analyses also reveal low protein abundance variations, suggesting the single-cell proteome profiles are highly stable for the cells cultured under identical conditions.

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59 BASIC BIOLOGICAL SCIENCESlab-on-a-chipmass spectrometryproteomeproteomics

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Woo, Jongmin (ORCID:0000000155699304), Williams, Sarah M., Markillie, Lye Meng (ORCID:0000000182754882), Feng, Song (ORCID:0000000339839009), Tsai, Chia-Feng, Aguilera-Vazquez, Victor, Sontag, Ryan L., Moore, Ronald J. (ORCID:0000000328062855), Hu, Dehong (ORCID:0000000239742963), Mehta, Hardeep S., Cantlon-Bruce, Joshua, Liu, Tao (ORCID:0000000195296550), Adkins, Joshua N. (ORCID:0000000303990700), Smith, Richard D. (ORCID:0000000223812349), Clair, Geremy C. (ORCID:0000000304788520), Pasa-Tolic, Ljiljana (ORCID:0000000198535457), Zhu, Ying (ORCID:0000000254160566). 2021-10-29. High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip. https://doi.org/10.1038/s41467-021-26514-2

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