Application of mass spectrometry to proteomic analysis of tissue is a highly desirable approach to discovery of disease biomarkers due to a direct correlation of findings to tissue/disease histology and in many respects obviating the need for model systems of disease. Both frozen and formalin-fixed, paraffin-embedded (FFPE) tissue can be interrogated; however, worldwide access to vastly larger numbers of highly characterized FFPE tissue collections derived from both human and model organisms makes this form of tissue more advantageous. Here, an approach to large-scale, global proteomic analysis of FFPE tissue is described that can be employed to discover differentially expressed proteins between different histological tissue types and thus discover novel protein biomarkers of disease.