Detection of >400 CD Biomarkers and Pathway Proteins in Single Immune Cells by Cyclic MIST for Single-Cell Proteomic Studies

The identification and characterization of immune cell subpopulations is critical to reveal cell development throughout life and immune responses to environmental factors. Next-generation sequencing technologies have dramatically advanced single-cell genomics and transcriptomics for immune cell classification. However, gene expression is often not correlated with protein expression, and immunotyping is mostly accepted in the protein format. Current single-cell proteomic technologies are either limited in multiplex capacity or not sensitive enough to detect the critical functional proteins. Herein we present a single cells cyclic multiplex in situ tagging (CycMIST) technology to simultaneously measure >400 proteins, a scale of >10 times than similar technologies. Such an ultra-high multiplexity is achieved by reiterative staining of the single cells coupled with a MIST array for detection. This technology has been thoroughly validated through comparison with flow cytometry and fluorescence immunostaining techniques. Both peripheral blood mononuclear cells (PBMCs) and T cells are analyzed by the CycMIST technology, and almost the entire spectrum of cluster of differentiation (CD) surface markers have been measured. The landscape of fluctuation of CD protein expression in single cells has been uncovered by our technology. Further study found T cell activation signatures and the protein-protein networks. This study represents the highest multiplexity of single immune cell marker measurement targeting functional proteins. With additional information from intracellular proteins of the same single cells, our technology can potentially facilitate mechanistic studies of immune responses under various disease conditions.