High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip
Year: 2021
Type: article
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.
Source: Nature Communications
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: gold
Estimated APC paid: $5,560
Funders: U.S. Department of Energy, U.S. Department of Health and Human Services, National Institutes of Health, Office of Science, National Heart, Lung, and Blood Institute, National Cancer Institute, National Institute of General Medical Sciences, Biological and Environmental Research, Laboratory Directed Research and Development, Pacific Northwest National Laboratory
Awards: G1202769797, G125460123, G138090931, G2506945562, G2510423872, G263956163, G2820049545, G2988778103, G3289159562, G3508161151, G3563651241, Research Center for Spatiotemporal Lung Imaging and Omics, G4462378477, G5321382714, G5373841109, A Research Resource for Ultra-sensitive High Throughput Proteomics, G6243784844, Center for Lung Development Imaging and Omics, G704717881, PNNL Proteome Characterization Center, G7562546725, G7673727097, G7913850483, G8135867528, G821644656, G8814379408, G9000182343
OpenAlex ID: W3209470205