Single-Cell Protein Assays in Context: From 2D to 3D and In Situ Analysis

Single-cell proteomics (scP) is a crucial complement to transcriptomics, offering deeper insight into cellular heterogeneity, disease mechanisms, and therapeutic vulnerabilities in samples such as 2D cell culture, 3D models, and patient tissue. While transcriptomics enables high-throughput gene expression characterization, RNA levels frequently do not correlate with protein levels even in the same cell. Furthermore, protein isoforms, posttranslational modifications, and complexes are missed by transcriptomic analyses. This review explores modern scP technologies, including flow and mass cytometry, single-cell mass spectrometry, immunohistochemistry, cyclic imaging, and imaging mass cytometry applied to both dissociated and spatially preserved samples. We emphasize applying these techniques to organ-on-a-chip, organoids, spheroids, and intact tissues, highlighting advances in spatial resolution and multiplexing. We also discuss the trade-offs between throughput, spatial fidelity, and protein selectivity across platforms. Finally, we identify key measurement gaps, suggesting future directions toward spatially resolved scP clinical translation.