High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip
| Autor: | Richard D. Smith, Hardeep S. Mehta, Chai-Feng Tsai, Song Feng, Tao Liu, Dehong Hu, Joshua N. Adkins, Ljiljana Paša-Tolić, Ronald J. Moore, Victor Aguilera-Vazquez, Ying Zhu, Joshua Cantlon-Bruce, Lye Meng Markillie, Jongmin Woo, Ryan L. Sontag, Sarah M. Williams, Geremy Clair |
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| Rok vydání: | 2021 |
| Předmět: |
Proteomics
Low protein Proteome Science General Physics and Astronomy Computational biology Tandem mass tag Article General Biochemistry Genetics and Molecular Biology Cell Line Specimen Handling Workflow Mice Tandem Mass Spectrometry Lab-On-A-Chip Devices Animals Sample preparation Author Correction Throughput (business) Multidisciplinary Lab-on-a-chip Mass spectrometry Sequence Analysis RNA Chemistry Proteins Reproducibility of Results Robustness (evolution) Equipment Design General Chemistry Chip Nanostructures RAW 264.7 Cells Single-Cell Analysis Biomarkers |
| Zdroj: | Nature Communications, Vol 12, Iss 1, Pp 1-11 (2021) Nature Communications |
| ISSN: | 2041-1723 |
| Popis: | 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 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. Single-cell proteomics is an emerging technology but protein coverage, throughput and quantitation accuracy are often still insufficient. Here, the authors develop a nested nanowell chip that improves protein recovery, throughput and robustness of isobaric labeling-based quantitative single-cell proteomics. |
| Databáze: | OpenAIRE |
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