Benchmarking sample preservation strategies for clinical single-cell proteomics

Single-cell proteomics (SCP) enables direct characterization of cellular states at proteome resolution but remains limited in clinical studies due to challenges in sample collection, storage, and transportation. Here, we systematically evaluated preservation strategies for SCP by comparing fresh, cryopreserved, and paraformaldehyde-fixed cells using HeLa cells as a benchmark model (n = 159 cells). Both strategies maintained comparable proteome coverage, but fixation altered protein abundance, whereas cryopreservation largely preserved the proteomic profile. To assess the translational applicability of this workflow, we analyzed single peripheral blood mononuclear cells (PBMCs) from one healthy donor, comparing freshly isolated cells (n = 77) with cells subjected to cryopreservation followed by extended-term storage (n = 75). In PBMCs, cryopreservation did not significantly affect protein group identifications, cellular morphology, proteome coverage, or global proteomic landscape. Together, these results demonstrate that cryopreservation provides a feasible strategy for preserving primary immune cells for downstream SCP under the conditions evaluated here, while comparative preservation strategies using single HeLa cells highlight the advantages and challenges of fixation and cryopreservation for future clinical applications.