Abstract
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 alpha6, 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.
Highlights
Cultivated vascular smooth muscle cells (SMCs) were surface-labeled with CyDyes followed by biotinylation
The DIGE/Biotin Labeling Procedure—We attempted to combine DIGE and biotin labeling of membrane proteins based on the principle that both techniques use an N-hydroxysulfosuccinimide (NHS) ester group to react with the -amine of lysines
Because CyDye DIGE fluor minimal dyes are designed to ensure the dyes are limiting in the reaction [12], biotin can target the remaining free lysines, the stoichiometry has to be kept low during primary amine derivatization so as not to compromise trypsinolysis
Summary
Cultivated vascular smooth muscle cells (SMCs) were surface-labeled with CyDyes followed by biotinylation. The cell adhesion molecule integrin ␣V has low intensity in the total protein stain but is strongly CyDye labeled (Fig. 5B).
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