nanoPOTS LC-MS for spatial single cell proteome (SSCP) analysis v1

State-of-the-art omics and imaging technologies for transcriptome, epigenome, and proteome measurements at the single-cell level have been successfully demonstrated and integrated into large tissue mapping consortia (e.g., HuBMAP, SenNet). However, current approaches for probing spatial distribution of the proteome typically rely on the use of antibodies, which limits multiplexing and requires a priori knowledge of protein targets. In this protocol, we describe LC-MS workflow for unbiased, deep mapping of the proteome, enabling the characterization of proteins within tissues, including senescent cells and their surrounding microenvironment. Following precise sample collection at high spatial resolution (5–10 μm), collected tissue samples are subjected to nanoscale proteomic processing and analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-MS/MS). This approach allows global cellular and subcellular proteomic profiling, supporting the exploration of protein dynamics at organellar scales. Here, we describe the LC-MS steps applied to mouse tissue samples to facilitate unbiased spatial single-cell proteomics (SSCP).