Integrated membrane protein analysis of mature and embryonic stem cell-derived smooth muscle cells using a novel combination of CyDye/biotin labeling.
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.
Sidibe
Anissa
A
Cardiovascular Division, King's College London School of Medicine, Kings College London, University of London, SE5 9NU London, United Kingdom.
Yin
Xiaoke
X
Tarelli
Edward
E
Xiao
Qingzhong
Q
Zampetaki
Anna
A
Xu
Qingbo
Q
Mayr
Manuel
M
D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin / Q000378:metabolism*; D004396:Coloring Agents / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D053595:Embryonic Stem Cells / Q000737:chemistry* / Q000166:cytology; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D032389:Myocytes, Smooth Muscle / Q000737:chemistry* / Q000166:cytology*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*
D004396:Coloring Agents; D008565:Membrane Proteins; D010455:Peptides; D011956:Receptors, Cell Surface; D001710:Biotin