High-Throughput Single-Cell Proteomics of In Vivo Cells.

Single-cell mass spectrometry-based proteomics (SCP) can resolve cellular heterogeneity in complex biological systems and provide a system-level view of the proteome of each cell. Major advancements in SCP methodologies have been introduced in recent years, providing highly sensitive sample preparation methods and mass spectrometric technologies. However, most studies present limited throughput and mainly focus on the analysis of cultured cells. To enhance the depth, accuracy, and throughput of SCP for tumor analysis, we developed an automated, high-throughput pipeline that enables the analysis of 1536 single cells in a single experiment. This approach integrates low-volume sample preparation, automated sample purification, and LC-MS analysis with the Slice-PASEF method. Integration of these methodologies into a streamlined pipeline led to a robust and reproducible identification of more than 3000 proteins per cell. We applied this pipeline to analyze tumor macrophages in a murine lung metastasis model. We identified over 1700 proteins per cell, including key macrophage markers and more than 500 differentially expressed proteins between tumor and control macrophages. PCA analysis successfully separated these populations, revealing the utility of SCP in capturing biologically relevant signals in the tumor microenvironment. Our results demonstrate a robust and scalable pipeline poised to advance single-cell proteomics in cancer research.