High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip
Jongmin Jacob Woo, Sarah Williams, Lye Meng Markillie, Song Feng, Chia‐Feng Tsai, Victor Aguilera-Vazquez, Ryan Sontag, Ronald Moore, Dehong Hu, Hardeep Mehta, Joshua Cantlon-Bruce, Tao Liu, Joshua Adkins, Richard Smith, Gérémy Clair, Ljiljana Paša‐Tolić, Ying Zhu
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.