We have previously demonstrated native nano-desorption electrospray ionization (nano-DESI) mass spectrometry imaging of proteins and protein complexes in thin tissue section of rat kidney. Here, we demonstrate the integration of travelling wave ion mobility spectrometry (TWIMS) into the native nano-DESI MSI workflow. The benefits of TWIMS are twofold: Firstly, arrival time filtering allows subtraction of chemical noise and the resulting ion images show improved specificity. Secondly, the incorporation of TWIMS enables the calculation of collision cross sections, and thus a measure of protein structure, directly from the imaging dataset. Our results show good agreement between the collision cross sections determined from nano-DESI, which requires the use of a heated inlet, and those determined experimentally from liquid extraction surface analysis (LESA) with an ambient temperature inlet, and those available in the literature. Ion images and collision cross sections are presented for a range of proteins and protein assemblies with molecular weights of up to 42.6 kDa.