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Laser capture microdissection coupled with mass spectrometry enables comprehensive proteomic profiling of selected tissue regions, but typically does not support cell-type-specific proteomic analysis. We present\n                  <jats:italic>NicheProt<\/jats:italic>\n                  , a 3D optical microscopy-guided, photobleaching-mediated cell barcoding approach for isolating intact specific cell types from defined microanatomical tissue compartments or niches. Using sequential bottom-up proteomic analysis, we defined two distinct phenotypes of CD11c\u207a dendritic cells based on their spatial locations in the inflamed mouse spleen. These two compartment-specific dendritic cell populations were characterized by proteomic signatures differing in the levels of 54 proteins. 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