In-depth and high-throughput single-cell proteomics workflow defines function subtypes

The cell, the fundamental unit of life, is crucial for understanding the molecular mechanisms and functions of organisms in both healthy and diseased states. Mass spectrometry-based single‐cell proteomics aims to characterize biological functions and heterogeneity at the level of proteins in an unbiased manner. Despite its clear potential, single-cell proteomics faces formidable challenges in sensitivity and throughput.
Here we develop a single-cell workflow for consistent, high-throughput and in-depth proteomic data acquisition. Our approach integrates an optimized oil-free workflow on a nanoliter dispensing device (cellenONE) with the Orbitrap Astral mass spectrometer (Thermo Scientific), effectively streamlining the proteomic acquisition process. By implementing the Whisper Zoom technology (Evosep), we increased the throughput to 80 or 120 samples per day (SPD). We also optimized a data-independent acquisition method with variable windows to enhance proteomic depth and quantitative consistency. This approach allowed us to achieved a proteomic depth of over 3,000 to 5,000 protein groups depending on cell size. The integration of multiplex-DIA (mDIA) can further multiply throughput by at least two-fold to 160 or 240 cells per day. Compared to single-cell RNA sequencing, single-cell proteomics exhibits lower variability and enables the definition of various cell states.
We applied this refined workflow to a range of biological questions. For instance, in the study of melanoma single cells, we successfully characterized the proteomic distinctions between treatment resistant "persister cells" and a control population of cells. We achieved a proteomic depth of about 5,000 protein groups with high data completeness, even covering many important transcription factors. We identified elevated signatures like epithelial-mesenchymal transition, glycolysis metabolism and hypoxia in the persister cells, compared to cell cycle and DNA repair terms in the control melanoma cells.
Our study demonstrates that single-cell proteomics can be performed with high throughput and significant proteomic depth, a depth that was state of the art for bulk proteomes not long ago. We also highlight the potential of single-cell proteomics in addressing complex biological questions and advancing our understanding of cellular heterogeneity.