Single-cell proteomics of saliva enables noninvasive cell profiling in oral cancer

Single-cell proteomics based on mass spectrometry (SCP) has the potential to resolve cellular heterogeneity in complex microenvironments without requiring prior antibody labeling, yet its application to clinically accessible biofluids remains limited. Here, we established a high-coverage, label-free SCP workflow capable of quantifying up to 5,400 protein groups per cell, as benchmarked using single fresh and cryopreserved HeLa cells. We then applied this workflow to noninvasive saliva and tear fluid samples from healthy donors (n = 398 cells), identifying up to 1,000 and 600 protein groups per cell, respectively. Extending the approach to cryopreserved salivary CD3⁺ T cells from patients with oral squamous cell carcinoma (OSCC; n = 526 cells), we quantified up to 1,600 protein groups per cell and identified proteomic cell states associated with immune remodeling. Furthermore, exploratory trajectory analyses suggested continuous proteomic state transitions that were consistent with publicly available single-cell transcriptomic datasets. Together, these results establish a workflow for single-cell proteomic profiling of clinically accessible and noninvasive biofluids and provide a framework for investigating cellular heterogeneity in translational and clinical research.