Cultivated vascular smooth muscle cells (SMCs) were surface-labelled with Cy-dyes followed by biotinylation. After enrichment on avidin columns, proteins were separated on large format gradient gels by SDS-PAGE. A comparison between Cydye- tagged and non-tagged gel bands revealed a substantial increase of protein identifications from membrane, membrane-associated and extracellular matrix proteins with a corresponding reduction in co-purified intracellular proteins. Notably, the majority of identified proteins were involved in cellular adhesion processes. To demonstrate the quantitative potential of this platform, we performed a comparison between mature and embryonic stem cell-derived smooth muscle cells (esSMCs) and identified the membrane proteins E-cadherin, integrin alpha 6 and CD98 (4F2) to be significantly upregulated in esSMCs suggesting that SMCs derived from embryonic stem cells maintain characteristics of their embryonic stem cell origin (ESC). This was subsequently confirmed by RT-PCR: despite expressing a panel of smooth muscle markers (SMA, Calponin and SM22), esSMCs remained positive for markers of stem cell pluripotency (Oct 4, Nanog and Rex 1). In summary, we describe a novel strategy for the profiling of cell membrane proteins. The procedure combines DIGE technology with biotin/avidin labelling to discriminate membrane and membrane-associated proteins from intracellular contaminants by fluorescent tagging and permits semi-quantitative differential expression analysis of membrane proteins.