Tissue Top-down microproteomic was performed on 3 brain regions. 156 references proteins from different cellular compartments were characterized. Moreover, 11 Alternative proteins issued from alternative open reading frames (AltORF) were identified and were relied to the brain regions and associated to the function of their reference proteins. Some proteins display specific post-translational modifications profiles or truncation linked the brain regions and their functions. Systemic biology performed on microproteome identified in each region allowed to associate sub-networks with functional physiology of each brain region. Back correlation of the local extracted and identified microproteome with tissue cellular localization was then performed by MALDI mass spectrometry imaging. 40 proteins including 4 Altproteins have been back correlated in MALDI MSI and are in line with their tissue extracted region and physiological function. Taken together, we established the molecular physiome of 3 rat brain regions through reference and hidden proteome characterization.