Cell Biochemistry and Biophysics

, Volume 53, Issue 3, pp 135–143

Limitations of the Colloidal Silica Method in Mapping the Endothelial Plasma Membrane Proteome of the Mouse Heart

  • Selvam Arjunan
  • Michael Reinartz
  • Barbara Emde
  • Klaus Zanger
  • Jürgen Schrader
Original Paper

DOI: 10.1007/s12013-009-9045-8

Cite this article as:
Arjunan, S., Reinartz, M., Emde, B. et al. Cell Biochem Biophys (2009) 53: 135. doi:10.1007/s12013-009-9045-8

Abstract

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.

Keywords

Colloidal silicaEndothelial membrane proteomeTransmission electron microscopy (TEM)Mass spectrometry (MS)

Supplementary material

12013_2009_9045_MOESM1_ESM.doc (410 kb)
(DOC 410 kb)

Copyright information

© Humana Press Inc. 2009

Authors and Affiliations

  • Selvam Arjunan
    • 1
  • Michael Reinartz
    • 1
  • Barbara Emde
    • 1
  • Klaus Zanger
    • 2
  • Jürgen Schrader
    • 1
  1. 1.Department of Cardiovascular PhysiologyHeinrich-Heine-University DüsseldorfDüsseldorfGermany
  2. 2.Department of Anatomy IIHeinrich-Heine-University DüsseldorfDüsseldorfGermany