Acetonitrile-based Proteomics of FACS-sorted Immune Cells Reveals Cell-type-specific Proteomes and Marker-gradient-resolved States

In this work, we develop and systematically evaluated an optimized sample preparation workflow based on acetonitrile (ACN) lysis and digestion, specifically designed for proteomic analysis of FACS-sorted cells. We demonstrate its utility and exploratory potential of this method through two case studies. Firstly, we profiled ATMs to validate the utility of the approach. ATMs are key regulators of tissue homeostasis, yet high-quality diet-induced proteomic data for this cell population remain scarce. Integrative comparison with scRNA-seq data revealed that our strategy offers superior sensitivity in detecting stimulus-induced changes within the same cell type. Furthermore, stratified proteomic analysis of PBMCs from colorectal cancer patients indicated continuous phenotypic classifications not fully captured by single-cell transcriptomics, highlighting the potential for in-depth characterization of protein and functional heterogeneity in immune cell populations. Collectively, this work establishes a versatile and powerful proteomic framework that bridges a critical technical gap, enabling high-depth, quantitative, and reproducible proteomic profiling of rare FACS-isolated cells, and providing a robust, scalable platform for both fundamental and translational research.