Extending single-cell proteomics from HeLa to small primary human immune cells

Abstract Single-cell proteomics (SCP) holds the promise of decoding cellular heterogeneity at the functional level, yet achieving deep and reproducible proteome coverage from individual cells has remained a formidable challenge. Here, we establish a high-sensitivity, label-free SCP platform that extends current depth and reproducibility. By integrating optimized low-input sample processing, refined liquid chromatography, and the Orbitrap Astral Zoom mass spectrometer, our approach routinely quantifies up to 7000 proteins per individual HeLa cell at over 100 samples-per-day (SPD), capturing thousands of low-abundance proteins that eluded prior SCP studies. Applied to very small human peripheral blood mononuclear cells (PBMCs), we identify up to 4000 proteins per cell, including key markers that distinguish monocytes, T cells, and activated lymphocytes within heterogeneous populations, underscoring that single-cell proteomics can now directly elucidate clinically relevant primary samples with both depth and precision.