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 would provide more direct information on cellular function phenotypes and complement transcriptomics measurements. However, single-cell proteomics (scProteomics) is still immature and confronts technical challenges, including limited proteome coverage, poor reproducibility, as well as low throughput. Here we describe a nested nanowell (N2) chip to dramatically improve protein recovery, operation robustness, and processing throughput for isobaric-labeling-based scProteomics workflow. The N2 chip allows reducing cell digestion volume to <30 nL and increasing processing capacity to > 240 single cells in one microchip. In the analysis of ~100 individual cells from three different cell lines, we demonstrate the N2 chip-based scProteomics platform can robustly quantify ~1500 proteins and reveal functional differences. Our analysis also reveals low protein abundance variations (median CVs < 16.3%), highlighting the utility of such measurements, and also suggesting the single-cell proteome is highly stable for the cells cultured under identical conditions.

A Data-Dependent Acquisition Ladder for Ultrasensitive (Neuro)Proteomics

“…Continuous advances at multiple stages of the proteomic workflow raise the possibility of further sensitivity improvements. Technologies enabling the isolation and handling of miniscule amounts of proteins with reduced loss, for example, by in vivo microsampling 16 , nanoPOTS 9 , and ScoPE 10 offer viable solutions for biopsies and limited populations of cells, including single cells. Detection of 428 proteins from protein amounts estimating ~10 neurons in this study, including many with important biological functions in homeostasis and disease, marks another technological leap in ultrasensitive proteomics, expanding the bioanalytical toolbox of neuroscience.…”

“…8 Recent nanoPOTS (N2) arrays analyzing 100 pg of tryptic protein digest identified ~1,300 proteins from single murine cells. 9 The automated single cell proteomics (SCoPE2) workflow enhanced throughput to ~200 single cells in 24 h using a refined nanoLC system, while reporting on ~1,000 proteins per cell. 10 These experiments deepened the detectable coverage of the single cell proteome, albeit at the expense of long experimental times (~2–5 h per analysis).…”

An automated workflow for multiplexed single-cell proteomics sample preparation at unprecedented sensitivity

“…Despite being acquired on different instruments, our setup and the carrier reduction vastly improves S/N reporter values compared to 7-15 S/N of the nanoPOTS or N2. 6,8,19 Interestingly, TMTpro experiments with and without the carrier resulted in higher S/N of the single cell channels compared to the TMT10-plex (Fig. 2b).…”

“…Previously, most nanoPOTS approaches relied on home built robotic dispensing, which was overcome with the nested nanoPOTS (N2). 8 Here the cellenONE® a commercial liquid dispensing instrument is used for both cell isolation and single cell preparation on the N2 substrate to further reduce reaction volume down to ~20 nL. Additionally, the N2 overcomes the need for manual combination of TMTmultiplexed sets using sparsely distributed arrays of single cell reaction sites, which can be unified with a microliter droplet on top of each sample set.…”

“…The authors demonstrated the reproducible analysis of TMT-multiplexed single cells in conjunction with an abundant 10 ng carrier sample transferring the combined sample to a vial or their custom built nanoPOTS autosampler. 8,9 We here describe a highly versatile and automated workflow for both label-free and isobaric multiplexed single cell proteomics sample preparation at unprecedented sensitivity. We introduce the proteoCHIP encompassing sixteen nanowells for cross-contamination-free simultaneous processing of up to 192 single cells per chip.…”