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Originally published In Press as doi:10.1074/mcp.M600433-MCP200 on July 11, 2007.
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Molecular & Cellular Proteomics 6:1788-1797, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Integrated Membrane Protein Analysis of Mature and Embryonic Stem Cell-derived Smooth Muscle Cells Using a Novel Combination of CyDye/Biotin Labeling *,S

Anissa Sidibe{ddagger}, Xiaoke Yin{ddagger}, Edward Tarelli§, Qingzhong Xiao{ddagger}, Anna Zampetaki{ddagger}, Qingbo Xu{ddagger} and Manuel Mayr{ddagger},¶

From the {ddagger} Cardiovascular Division, King's College London School of Medicine, Kings College London, University of London, SE5 9NU London, United Kingdom and § Medical Biomics Center, St. George's, University of London, SW17 0RE London, United Kingdom

Cultivated vascular smooth muscle cells (SMCs) were surface-labeled with CyDyes 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 up-regulated in esSMCs suggesting that SMCs derived from embryonic stem cells maintain characteristics of their embryonic stem cell origin. This was subsequently confirmed by RT-PCR: despite expressing a panel of smooth muscle markers (calponin, Sm22, and aortic smooth muscle actin), esSMCs remained positive for markers of stem cell pluripotency (Oct4, Nanog, and Rex1). In summary, we describe a novel strategy for the profiling of cell membrane proteins. The procedure combines DIGE technology with biotin/avidin labeling to discriminate membrane and membrane-associated proteins from intracellular contaminants by fluorescence tagging and permits semiquantitative differential expression analysis of membrane proteins.

To whom correspondence should be addressed: Cardiovascular Division, The James Black Centre, King's College London School of Medicine, King's College London, University of London, 125 Coldharbour Lane, London SE5 9NU, UK. Tel.: 44-20-7848-5238; Fax: 44-20-7848-5296; E-mail: manuel.mayr{at}kcl.ac.uk


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