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Glycoproteomic analysis of the secretome of human endothelial cells.

Reputable Mentor II
Reputable Mentor II
Yin X, Bern M, Xing Q, Ho J, Viner R, Mayr M.
Mol Cell Proteomics. 2013 Jan 23.
Previous proteomics studies have partially unravelled the complexity of endothelial protein secretion, but did not investigate glycosylation, a key modification on secreted and membrane proteins for cell communication. 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. Apart from 123 secreted proteins, the secretome was particularly rich in membrane proteins. Glycopeptides were enriched by ZIC-HILIC resins and were either treated with PNGase F and H(2)(18)O or directly analysed using a recently developed HCD-ETD workflow for a hybrid linear ion trap-orbitrap MS. After deglycosylation with PNGase F in the presence of H(2)(18)O, 123 unique peptides displayed (18)O-deamidation of asparagine, corresponding to 86 proteins with a total of 121 glycosylation sites. Direct glycopeptides analysis by HCD-ETD identified 131 glycopeptides from 59 proteins and 118 glycosylation sites, of which 41 were known, 51 were predicted and 26 were novel. Two methods were compared: alternating HCD-ETD and HCD-product dependent ETD. The former detected predominantly high intensity, multiply charged glycopeptides while the latter preferentially selected precursors with complex / hybrid glycans for fragmentation. Validation was performed by glycoprotein enrichment and analysing the input, the flow through and the bound fraction. This study represents the most comprehensive characterisation of endothelial protein secretion to date and demonstrates the potential of new HCD-ETD workflows for determining the glycosylation status in complex biological samples.
King’s British Heart Foundation Centre, King’s College London, London, UK
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