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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, Xiaoke Yin, Edward Tarelli, Qingzhong Xiao, Anna Zampetaki, Qingbo Xu and Manuel Mayr^,¶

From the 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 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.

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				M. Mayr, A. Zampetaki, A. Sidibe, U. Mayr, X. Yin, A. I. De Souza, Y.-L. Chung, B. Madhu, P. H. Quax, Y. Hu, et al. Proteomic and Metabolomic Analysis of Smooth Muscle Cells Derived From the Arterial Media and Adventitial Progenitors of Apolipoprotein E-Deficient Mice Circ. Res., May 9, 2008; 102(9): 1046 - 1056. [Abstract] [Full Text] [PDF]