Multiplexed single-cell proteomics using SCoPE2

Abstract

Many biological systems are composed of diverse single cells. This diversity necessitates functional and molecular single-cell analysis. Single-cell protein analysis has long relied on affinity reagents, but emerging mass-spectrometry methods (either label-free or multiplexed) have enabled quantifying over 1,000 proteins per cell while simultaneously increasing the specificity of protein quantification. Isobaric carrier based multiplexed single-cell proteomics is a scalable, reliable, and cost-effective method that can be fully automated and implemented on widely available equipment. It uses inexpensive reagents and is applicable to any sample that can be processed to a single-cell suspension. Here we describe an automated Single Cell ProtEomics (SCoPE2) workflow that allows analyzing about 200 single cells per 24 hours using only standard commercial equipment. We emphasize experimental steps and benchmarks required for achieving quantitative protein analysis.

Exploring functional protein covariation across single cells using nPOP

“…3a,b are mostly absent or very low, indicating that background noise is low for samples prepared with nPOP. The RI intensities for single cells also show that, as expected and previously observed 39 , peptides from Hela cells are more abundant than peptides from U937 cells, likely reflecting the different cell sizes.…”

“…Similar to previous analysis 38,39,46,47 , the bulk samples clustered with the corresponding single cells.…”

“…Thus, numerous methods have been developed for preparing sub-microgram protein samples [22][23][24][25] and single-cell samples [26][27][28][29][30][31][32][33][34][35] . To enable some degree of parallel processing, some methods have been automated using multiwell plates 32,35,36 . Preparing small samples often uses sophisticated custom-made equipment [27][28][29][30][31] .…”

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.…”

“…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).…”