Multiplexed Deep Visual Proteomics Unveils Spatial Heterogeneity and Rare Endocrine States in Human Adult Pancreatic Islets

Abstract

Pancreatic islets are highly specialized tissue compartments that regulate metabolism, and their dysfunction contributes to diseases such as prediabetes and diabetes. Characterizing islet morphology and cell plasticity is essential for understanding these pathophysiological states, yet high-resolution spatial proteomics remains challenging due to the islets small size and cellular complexity. Here, we present multiplexed deep visual proteomics (mxDVP), an approach that integrates high-plex imaging with ultra-sensitive mass spectrometry to achieve deep spatial proteome profiling of defined cell types in tissue sections. Enhanced segmentation, semi- automated annotation, and optimized laser microdissection enable the enrichment of rare endocrine populations that are often overlooked in single-cell analyses. mxDVP achieves deep proteome coverage of >6,000 proteins from as few as 100 cells, including low-abundance transcription factors critical for endocrine cell fate determination. By profiling over 864,000 human pancreatic cells, we identify 12 endocrine subtypes, including polyhormonal hybrids, revealing previously unrecognized islet heterogeneity, metabolic regulation, and cellular adaptability.