Overcoming Preservation Challenges to Enable Single-Cell Proteomics of Fixed Cells and Tissue Samples with Retained Proteome Integrity

The ability to assay the molecular composition of biological systems with single-cell resolution has revolutionized our understanding of tissue heterogeneity and function. Recent advances in single-cell proteomics (SCP) now enable the unbiased quantification of the proteome to a depth of several thousand proteins across hundreds of cells. Yet, broader adoption beyond specialized groups remains limited due to the need for specific equipment and expertise. A major challenge in making these analyses more broadly available is sample preservation for the transport of biological material to SCP-capable facilities. To address this issue and provide practical solutions, we first evaluated various cell preservation methods from monolayer culture samples, then tested our optimized methodology on both cultured cells and, for the first time, preserved animal tissue from an <i>in vivo</i> mouse model. Our findings highlight that the feasibility of SCP analyses in preserved tissues is more likely to be successful, significantly expanding its current applicability. By optimizing upstream processing, our approach enables robust single-cell proteome analysis of both cells and tissues, making SCP more accessible to the wider scientific community. Ultimately, this advancement expands the potential applications of SCP, particularly in disciplines where analyzing rare or heterogeneous populations is beneficial.