Oli Hale (@olijhale.bsky.social)

Imaging of Protein Assemblies up to 231 kDa in Tissues with Nano-DESI Mass Spectrometry Understanding the distribution of proteins and their assemblies in tissues is a major challenge in spatial biology. Mass spectrometry imaging (MSI) with nanospray-desorption electrospray ionization (nano-DESI) has previously enabled detection, imaging, and identification of intact protein complexes directly from tissues, including protein assemblies and pathological protein–metal complexes in neurodegenerative disease. To date, nano-DESI MSI has been most effective for lower molecular weight (MW) complexes (<100 kDa), with an upper limit of 113 kDa. Here, we demonstrate nano-DESI at molecular weights up to 231 kDa, more than doubling the previous limit, by combining nano-DESI with a new mass spectrometer system architecture designed for higher MW analysis. Both mouse brain and rat kidney tissues were analyzed. Importantly, protein identification by native top-down MS was performed exclusively by use of nano-DESI. That is, complementary techniques for protein identification, such as liquid extraction surface analysis, were not necessary. Both homo- and heteromeric proteoform assemblies were identified in complex with endogenous small-molecule and metal ion cofactors. The developments lead the way to the analysis of larger oligomeric protein assemblies and protein complexes, cementing nano-DESI as a tool for structural biology, and with implications for molecular pathology and drug discovery. https://pubs.acs.org/doi/10.1021/acs.analchem.5c05767