High-Throughput Spatial Proteomics at Cellular and Subcellular Resolution via a Hanging Droplet Workflow

Formalin-fixed paraffin-embedded (FFPE) samples are essential for clinical research and spatial proteomics (SP) but are technically challenging to analyze by liquid-chromatography coupled mass-spectrometry (LC-MS) due to losses during protein extraction, evaporation during de crosslinking and low-throughput sample preparation methods. Here, we present a streamlined micro-FFPE proteomics sample preparation protocol for laser-capture micro-dissection (LMD) that enables scalable and high-throughput processing in 96-well format, completing the entire process from tissue lysis to tryptic peptide mixtures on LC-MS-ready Evotips in just 2-hours. Our method integrates whole-slide imaging, LMD, and ultra-sensitive narrow-window data-independent acquisition (nDIA)-MS and facilitates enhanced lysis, de crosslinking, and proteolytic digestion directly within Teflon-based EVO96 chips. By utilizing nano-wells and the hanging droplet concept, our approach significantly reduces surface adsorption, thereby improving processing efficiency for both higher- and very low-input FFPE samples. This enables quantification of >5,500 protein groups (PGs) by nDIA-MS from 50,000 µm² (∼250 cells) with robust performance maintained down to 1000 µm ² (∼5 cells) with ∼2,500 PGs identified. Application to a panel of breast cancer (BC) tissues precisely discerned BC subtypes and their tumor micro-environment (TME) based on differential MS-based protein abundance of biomarkers including TP53, HER2 and PGR. Individually excised subcellular regions (∼60 µm²) yielded ∼1,500 PGs, demonstrating reliable micro-scale spatial proteomics profiling approaching near-organelle resolution.