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

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.

Parallel measurement of transcriptomes and proteomes from same single cells using nanodroplet splitting

“…To enable efficient integration, we developed nanoSPLITS (nanodroplet SPlitting for Linked-multimodal Investigations of Trace Samples), a method capable of equally dividing nanoliter-scale cell lysates via two droplet microarrays and separately measuring them with RNA sequencing and mass spectrometry. NanoSPLITS builds on the nanoPOTS platform that allows for high-efficiency proteomic preparation of single cells by miniaturizing the assay volumes to nanoliter scale volumes 16,17 . We have previously demonstrated reaction miniaturization not only reduces non-specific adsorption-related sample losses, but also enhances enzymatic digestion kinetics.…”

Microfluidic Compartmentalization Platforms for Single Cell Analysis

“…For example, integration with Illumina next-generation sequencing (NGS) enables genetic profiling of the high-affinity, antigen-reactive antibodies. The use of nanowells in combination with nanodroplets has also contributed to the liquid chromatography-mass spectrometry (LC-MS) of single-cell proteomes [ 87 , 88 ]. Many reviews have been published describing the integrated systems and their wide variety of applications [ 16 , 28 ].…”

Three-dimensional feature matching improves coverage for single-cell proteomics based on ion mobility filtering

“…Recent advances in sample preparation and mass spectrometry facilitate unbiased single-cell proteomics (scProteomics) (Budnik et al, 2018;Cheung et al, 2021;Cong et al, 2020;Dou et al, 2019;Hartlmayr et al, 2021;Li et al, 2018;Schoof et al, 2021;Shao et al, 2018;Specht et al, 2021;Tsai et al, 2020;Williams et al, 2020;Woo et al, 2021;Zhu et al, 2018aZhu et al, , 2018bZhu et al, , 2019. Microfluidic sample processing devices and systems have improved protein digestion efficiency and minimized adsorptive sample losses (Hartlmayr et al, 2021;Li et al, 2018;Shao et al, 2018;Woo et al, 2021;Zhu et al, 2018aZhu et al, , 2018b. Tandem mass tag (TMT)-based isobaric labeling approaches (e.g., ScoPE-MS) have enabled multiplexed singlecell analysis in individual LC-MS runs (Budnik et al, 2018;Dou et al, 2019;Hartlmayr et al, 2021;Schoof et al, 2021;Specht et al, 2021;Tsai et al, 2020).…”

“…High-resolution MS analyzers combined with ion focusing devices, such as ion funnel, have increased detection sensitivity to the level where single molecules can be detected (Makarov and Denisov, 2009). State-of-the-art methodologies in scProteomics now can identify from $700 to $1,000 proteins from cultured single mammalian cells (e.g., HeLa) using label-free approaches (Cong et al, 2020(Cong et al, , 2021Williams et al, 2020;Zhu et al, 2018a) and from $750 to $1,500 proteins using TMT-labeling strategies (Budnik et al, 2018;Dou et al, 2019;Schoof et al, 2021;Specht et al, 2021;Tsai et al, 2020;Woo et al, 2021). Despite these advances, scProteomics remains immature, and technical challenges remain, including not only limited proteome depth and poor quantification performance but also low system robustness for large-scale single-cell studies.…”