Quantitative single-cell proteomics as a tool to characterize cellular hierarchies

Abstract

Large-scale single-cell analyses are of fundamental importance in order to capture biological heterogeneity within complex cell systems, but have largely been limited to RNA-based technologies. Here we present a comprehensive benchmarked experimental and computational workflow, which establishes global single-cell mass spectrometry-based proteomics as a tool for large-scale single-cell analyses. By exploiting a primary leukemia model system, we demonstrate both through pre-enrichment of cell populations and through a non-enriched unbiased approach that our workflow enables the exploration of cellular heterogeneity within this aberrant developmental hierarchy. Our approach is capable of consistently quantifying ~1000 proteins per cell across thousands of individual cells using limited instrument time. Furthermore, we develop a computational workflow (SCeptre) that effectively normalizes the data, integrates available FACS data and facilitates downstream analysis. The approach presented here lays a foundation for implementing global single-cell proteomics studies across the world.

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

“…S1d , the user can manually add an 8-µL droplet inside the hydrophilic ring to pool the TMT-labeled single-cell samples and transfer it into an autosampler vial for LC injection. Recently, Schoof et al 19 and Liang et al 20 have demonstrated the Opentrons OT-2 liquid handler can reliably pipette low-µL-scale solutions for preparing single-cell samples. Similarly, the TMT pooling step for the N2 chip could be automated with conventional LC systems using the OT-2 robot.…”

“…Although label-free approaches exhibit better quantification accuracy and higher dynamic range, their throughputs are limited, as each cell requires a >0.5 h-long LC-MS analysis. In the isobaric labeling approaches (e.g., tandem mass tags, TMT) 14 – 19 , single-cell digests are labeled with unique isobaric tags that are then pooled together for a multiplex LC-MS analysis. Importantly, the peptides originating from different single cells appear as a single MS1 peak.…”

The glycosylation in SARS-CoV-2 and its receptor ACE2

“… 99 , 206 – 208 Therefore, the methods for acquiring evident MS2 signals and the algorithms of parsing the MS2 data are necessary. 209 – 213…”

Scaling Up Single-Cell Proteomics

“…This increased appreciation of the need to perform single-cell protein measurements has stimulated the development of single-cell MS methods that can identify and quantify hundreds of proteins from single cells at an unprecedented scale ( 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ). These methods aim to achieve similar objectives, such as efficient delivery of peptides from single cells to the MS instruments via miniaturized sample preparation ( 1 ), but differ in the approaches used for achieving these objectives.…”

“…Methods for multiplexed single-cell proteomics have relied primarily on using isobaric mass tags, usually combined with the isobaric carrier approach ( 32 ). This approach was introduced by Single-Cell ProtEomics by MS (SCoPE-MS) ( 22 ) and has been incorporated in its second version SCoPE2 ( 21 ) and other highly similar methods ( 25 , 26 , 33 , 34 ). This approach has also allowed deep proteome quantification from small cancer samples ( 35 ) and increased sensitivity of thermal proteome profiling ( 36 ).…”