Navigating the Landscape of Cytometry-Based Single-Cell Proteomics: Quantification, Annotation, and Resources

Cytometry-based single-cell proteomics (CySCP) has emerged as a powerful tool for analyzing cellular heterogeneity at the protein level because of its ability to reveal dynamic cell states and response patterns through high-dimensional protein expression profiling in thousands of individual cells. However, detailed summaries of quantification, processing and analysis of CySCP data remain limited. This review provides comprehensive perspectives on CySCP, including quantification technologies, analysis pipelines, annotation strategies, and resource platforms. Specifically, first, the strengths and limitations of the detection platforms are discussed. Second, comprehensive data processing steps, including compensation, transformation, normalization, batch effect correction, signal cleaning, and doublets, debris or dead cells removal, are described in detail. Third, various strategies for cell type annotation, including manual gating, unsupervised clustering, supervised/semi-supervised classification, and fully automated approaches, are illustrated. Fourth, emerging CySCP databases, as critical resources for facilitating antibody validation, panel optimization, and open-access data sharing, are summarized. In summary, this review provides a comprehensive guide for the use of CySCP to obtain novel biological insights at the single-cell protein level.