Proteomic analysis of lipid microdomains from lipopolysaccharide-activated human endothelial cells

Aly Karsan*, Josip Blonder, Jennifer Law, Elisa Yaquian, David A. Lucas, Thomas P. Conrads, Timothy Veenstra

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

39 Scopus citations


The endothelium plays a critical role in orchestrating the inflammatory response seen during sepsis. Many of the inflammatory effects of Gram-negative sepsis are elicited by lipopolysaccharide (LPS), a glycolipid component of bacterial cell walls. Lipid-rich microdomains have been shown to concentrate components of the LPS signaling system. However, much remains to be learned about which proteins are constituents of lipid microdomains, and how these are regulated following cell activation. Progress in this area would be accelerated by employing global proteomic analyses, but the hydrophobicity of membrane proteins presents an analytical barrier to the effective application of such approaches. Herein, we describe a method to isolate detergent-resistant membranes from endothelial cells, and prepare these samples for proteomic analysis in a way that is compatible with subsequent separations and mass spectrometric (MS) analysis. In the application of these sample preparation and MS analyses, 358 proteins from the lipid-rich microdomains of LPS-activated endothelial cell membranes have been identified of which half are classified as membrane proteins by Gene Ontology. We also demonstrate that the sample preparation method used for solubilization and trypsin digestion of lipid-rich microdomains renders the membrane spanning sequences of transmembrane proteins accessible for endoproteolytic hydrolysis. This analysis sets the analytical foundation for an in-depth probing of LPS signaling in endothelial cells.

Original languageEnglish
Pages (from-to)349-357
Number of pages9
JournalJournal of Proteome Research
Issue number2
StatePublished - Mar 2005
Externally publishedYes


  • Caveolae
  • Endothelium
  • Inflammation
  • Lipid rafts
  • Lipid-rich microdomains
  • Lipopolysaccharide


Dive into the research topics of 'Proteomic analysis of lipid microdomains from lipopolysaccharide-activated human endothelial cells'. Together they form a unique fingerprint.

Cite this