PMID: 23345538

 

    Legend: Sugar

Title : Glycoproteomic analysis of the secretome of human endothelial cells

Abstract :
  1. Previous proteomics studies have partially unraveled the complexity of endothelial protein secretion but have not investigated glycosylation, a key modification of secreted and membrane proteins for cell communication
  2. In this study, human umbilical vein endothelial cells were kept in serum-free medium before activation by phorbol-12-myristate-13 acetate, a commonly used secretagogue that induces exocytosis of endothelial vesicles
  3. In addition to 123 secreted proteins , the secretome was particularly rich in membrane proteins
  4. Glycopeptides were enriched by zwitterionic hydrophilic interaction liquid chromatography resins and were either treated with PNGase F and H2(18)O or directly analyzed using a recently developed workflow combining higher-energy C-trap dissociation ( HCD ) with electron-transfer dissociation (ETD) for a hybrid linear ion trap-orbitrap mass spectrometer
  5. After deglycosylation with PNGase F in the presence of H2(18)O, 123 unique peptides displayed (18)O-deamidation of asparagine , corresponding to 86 proteins with a total of 121 glycosylation sites
  6. Direct glycopeptide analysis via HCD-ETD identified 131 glycopeptides from 59 proteins and 118 glycosylation sites , of which 41 were known, 51 were predicted, and 26 were novel
  7. Two methods were compared: alternating HCD-ETD and HCD-product-dependent ETD
  8. The former detected predominantly high-intensity, multiply charged glycopeptides , whereas the latter preferentially selected precursors with complex/hybrid glycans for fragmentation
  9. Validation was performed by means of glycoprotein enrichment and analysis of the input, the flow-through, and the bound fraction
  10. This study represents the most comprehensive characterization of endothelial protein secretion to date and demonstrates the potential of new HCD-ETD workflows for determining the glycosylation status of complex biological samples