Are we there yet? Assessing the readiness of single-cell proteomics to answer biological hypotheses

Single-cell measurements are crucial in investigating biological hypotheses that involve cell-to-cell heterogeneity. Bulk measurements, generated with hundreds or thousands of cells, represent sample averages and can obscure functionally important cell-to-cell differences. Mass-spectrometry-based single-cell proteomics (MS-based SCP) has seen a lot of work in method development to make it a viable investigative option. Improvements in sample preparation and methods for data acquisition have led to greatly-increased proteomic depth from a single cell (~3,000-5,000 proteins per cell). Our goal was to assess the readiness of SCP to participate in biological investigations, such as: identifying all subpopulations in a sample, even rare cells (cell annotation); reconstructing trajectories representing a continuous arc of development for a cell type of interest (developmental trajectory inference); and analyzing cells in relation to their location in a microenvironment (spatial mapping).We identified sample throughput as the primary limitation of SCP. Additionally, we identified important experimental design and data analysis considerations specific to the SCP data type, including: sample size, proteomic depth, batch effects, double dipping, assumptions of available analysis tools, and spatial resolution tradeoffs.