The formation of blood vessels (angio genesis) and of lymphatic vessels (lymphangiogenesis) actively contributes to cancer progression and inflam mation. Thus, there has been a quest for identifying the molecular mechanisms that control lymphatic and blood vessel formation and function. Membrane and extracellular matrix proteins can serve as suitable tar gets for imaging and/or therapeutic targeting;however, conventional proteomic technologies often fail to iden tify them systematically due to insolubility in water and low abundance of membrane proteins. To circumvent this problem, we applied a gel‐free proteomics meth odology termed two‐dimensional peptide mapping (2D‐ PM) to cultured blood vascular (BECs) and lymphatic (LECs) endothelial cells. 2D‐PM comprises biotinylation of surface‐accessible proteins, their selective en richment, separation by HPLC, and analysis by mass spectrometry. We identified 184 proteins that were specifically or predominantly expressed by LECs and 185 proteins specifically expressed by BECs, whereas 377 additional proteins were equally detected in both cell types. For representative proteins, the differential, lineage‐specific expression was confirmed by Western analyses of cultured cells and by differential immuno fluorescence analyses of tissue samples. Our results identify the surface‐accessible, vascular lineage‐specific proteome, and they also reveal 2D‐PM as a powerful technology for the large‐scale screening of lineage‐ specific protein expression.— Roesli, C., Mumprecht, V., Neri, D., Detmar, M. Identification of the surface‐ accessible, lineage‐specific vascular proteome by two dimensional peptide mapping. FASEB J. 22, 1933–1944 (2008)