Native Mass Spectrometry Imaging of Proteins and Protein Complexes by Nano-DESI

[Image: see text] Previously, we have demonstrated native mass spectrometry imaging (native MSI) in which the spatial distribution of proteins maintained in their native-like, folded conformations was determined using liquid extraction surface analysis (LESA). While providing an excellent testbed for proof of principle, the spatial resolution of LESA is currently limited for imaging primarily by the physical size of the sampling pipette tip. Here, we report the adoption of nanospray-desorption electrospray ionization (nano-DESI) for native MSI, delivering substantial improvements in resolution versus native LESA MSI. In addition, native nano-DESI may be used for location-targeted top–down proteomics analysis directly from tissue. Proteins, including a homodimeric complex not previously detected by native MSI, were identified through a combination of collisional activation, high-resolution MS and proton transfer charge reduction.