For investigating the molecular physiology and pathophysiology in organs the most exact data will be obtained, if not organ specific cell lines are analysed or the whole organ is homogenized, followed by the analysis of its biomolecules, but if the morphological organisation of the organ can be addressed and in the best case the composition of molecules in single cells of the target organ can be analysed. Laser capture microdissection (LCM) is a technique, which is enabling the selection of specific cells of a tissue for further analysis of their molecules. However, LCM is a time-consuming 2-dimensional technique and optimal results are only obtained, if the tissue is fixed e.g. by formalin. Especially for proteome analysis formalin fixation is reducing the number of identifiable proteins, which is an additional drawback. Recently, it was demonstrated that sampling of fresh-frozen (non-fixed) tissue with an infrared-laser is giving higher yields with respect to the absolute protein amount and number of identifiable proteins than conventional mechanical homogenization of tissues. In this study the applicability of the infrared laser tissue sampling for proteome analysis of different cell layers of murine intestine was investigated, using LC-MS/MS-based differential quantitative bottom-up proteomics. By laser ablation eight consecutive layers of the colon tissue were obtained and analyzed. Beside a clear distinguishability of protein profiles between ascending, descending and transversal colon, we identified in the different intestinal cell layers proteins, which are cell-specific as confirmed with data from the human protein atlas. Thus, for the first time the sampling directly from intact fresh-frozen tissue with 3-dimensional resolution is giving access to the different proteomes of the different cell layers of colon tissue.