Chemical maps of tissue samples provide important information on biological processes therein. Recently, advances in tissue imaging have been achieved using am-bient ionization techniques, such as desorption electrospray ionization mass spectrometry (DESI-MS), but such tech-niques have been almost exclusively confined to the mapping of lipids and metabolites. We report here the use of na-nospray desorption electrospray ionization (nanoDESI) al-lows us to image proteins in tissue samples in a label-free manner at atmospheric pressure with only minimum sample preparation. Multiply charged proteins with masses up to 15 kDa were successfully detected by nanoDESI using an LTQ Orbitrap mass spectrometer. In an adult mice brain section, expression of proteins including ubiquitin, β-thymosin, mye-lin basic protein, and hemoglobin were spatially mapped, and characterized. We also determined the location of meth-ylation on myelin basic protein. This imaging modality was further implemented to MYC-induced lymphomas. We ob-served an array of truncated proteins in the region where normal thymus cells were infiltrated by tumor cells, in con-trast to healthy tissue.