Arjunan Selvam, Reinartz Michael, Emde Barbara, Zanger Klaus, Schrader Jürgen
Department of Cardiovascular Physiology, Heinrich-Heine-University Düsseldorf, Universitätsstr. 1, 40225, Düsseldorf, Germany.
Cell Biochem Biophys. 2009;53(3):135-43. doi: 10.1007/s12013-009-9045-8.
The endothelial cell (EC) membrane is an important interface, which plays a crucial role in signal transduction. Our aim was to selectively purify luminal EC membrane proteins from the coronary vasculature of the isolated perfused mouse heart and analyze its composition with mass spectrometry (MS). To specifically label coronary ECs in the intact heart, the colloidal silica method was applied, which is based on the binding of positively charged colloidal silica to the surface of EC membranes. Transmission electron microscopy revealed the specific labeling of ECs of macro and microvessels. Two different methods of tissue homogenization (Teflon pestle and ultra blade) together with density centrifugation were used for membrane protein enrichment. Enrichment and purity was controlled by Western blot analysis using the EC-specific protein caveolin 1 and various intracellular marker proteins. The ultra blade method resulted in a tenfold enrichment of caveolin 1, while there was negligible contamination as judged by Western blot. However, protein yield was low and required pooling of ten hearts for MS. When enriched endothelial membrane proteins were digested with trypsin and analyzed by LC-MS, a total of 56 proteins could be identified, of which only 12 were membrane proteins. We conclude that coronary endothelial membranes can be conveniently labeled with colloidal silica. However, due to the ionic nature of interaction of colloidal silica with the EC membrane the shear rate required for cardiac homogenization resulted in a substantial loss of specificity.
内皮细胞膜是一个重要的界面，在信号转导中起关键作用。我们的目的是从离体灌注小鼠心脏的冠状血管中选择性纯化管腔内皮细胞膜蛋白，并用质谱（MS）分析其组成。为了在完整心脏中特异性标记冠状内皮细胞，应用了胶体二氧化硅方法，该方法基于带正电荷的胶体二氧化硅与内皮细胞膜表面的结合。透射电子显微镜显示了大血管和微血管内皮细胞的特异性标记。两种不同的组织匀浆方法（聚四氟乙烯杵和超刀片）与密度离心一起用于膜蛋白富集。使用内皮细胞特异性蛋白小窝蛋白1和各种细胞内标记蛋白通过蛋白质印迹分析来控制富集和纯度。超刀片方法使小窝蛋白1富集了10倍，而通过蛋白质印迹判断污染可忽略不计。然而，蛋白质产量低，需要合并10个心脏用于质谱分析。当用胰蛋白酶消化富集的内皮膜蛋白并通过液相色谱-质谱分析时，总共可以鉴定出56种蛋白质，其中只有12种是膜蛋白。我们得出结论，冠状内皮细胞膜可以方便地用胶体二氧化硅标记。然而，由于胶体二氧化硅与内皮细胞膜相互作用的离子性质，心脏匀浆所需的剪切速率导致特异性大量丧失。
