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

Jongmin Woo,Lye Meng Markillie,Joshua N Adkins,Sarah M Williams,Chia-Feng Tsai,Ryan L Sontag,Geremy C Clair,Hardeep S Mehta,Tao Liu,Ljiljana Pasa-Tolic,Richard D Smith,Song Feng,Dehong Hu,Joshua Cantlon-Bruce,Ying Zhu,Victor Aguilera-Vazquez,Ronald J Moore

doi:10.1038/s41467-021-26514-2

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.

Highlights

Global quantification of protein abundances in single cells could provide direct information on cellular phenotypes and complement transcriptomics measurements
The immune-cell-related markers, CD14, CD68, and CYBA (Uniprot protein name: CY24A_Human), are localized explicitly in macrophage cells in human lung tissues. These results demonstrated cell-type-specific surface markers can be effectively identified by combining scProteomics with subcellularlocalization information
We have developed a high-throughput and streamlined scProteomics sample preparation workflow based on nested nanoPOTS (N2) chips

Summary

Introduction

Global quantification of protein abundances in single cells could provide direct information on cellular phenotypes and complement transcriptomics measurements. We describe a nested nanoPOTS (N2) chip to improve protein recovery, operation robustness, and processing throughput for isobaric-labeling-based scProteomics workflow. 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. ScProteomics still lags behind single-cell transcriptomics in terms of coverage, measurement throughput, and quantitation accuracy[4]. In the label-free methods[5,6,7,8,9,10], single cells are individually processed and analyzed with liquid chromatography-mass spectrometry (LC-MS).

Methods

Results

Conclusion