P586Proteomic analysis of the secretome of activated human umbilical vein endothelial cells

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Abstract

Background

Previous studies have partially unravelled the complexity of endothelial protein secretion, but further studies are needed to achieve a more comprehensive characterisation of this important subproteome, in particular with regards to post-translational modifications such as glycosylation.

Methods and Results

Human umbilical vein endothelial cells (HUVECs) were kept in serum-free medium before activation by phorbol 12-myristate 13-acetate (PMA, 50nM) for 45 min. Proteins in the conditioned media were analysed by gel-LC-MS/MS using an LTQ Orbitrap XL mass spectrometer (Thermo Fisher). The glycoprotein-enriched fraction was analysed by a Q Exactive or Orbitrap Elite mass spectrometer (Thermo Fisher). In the conditioned medium of PMA-stimulated endothelial cells, 962 proteins were identified, significantly more than in previous proteomic studies investigating endothelial protein secretion either at baseline (Tunica et. al. 2009) or in response to mechanical stimulation (Burghoff et. al. 2011). Besides secreted proteins (n=135), the conditioned medium was particularly rich in membrane proteins (n=193). 77 proteins were membrane antigens, receptors or proteins related to cross-membrane transport, which is consistent with the known effects of PMA, a commonly used secretagogue that induces exocytosis of endothelial vesicles. Furthermore, glycoprotein enrichment enabled the identification of glycosylation sites as well as the sugar-chain composition of glycoproteins.

Conclusions

This study represents the most comprehensive characterisation of endothelial protein secretion to date and demonstrates the potential of mass spectrometry for determining their glycosylation status.

Conclusions

Reference: Burghoff S and Schrader J. Secretome of human endothelial cells under shear stress. J. Proteome Res., 2011; 10(3): 1160-1169. Tunica DG, Yin X, Sidibe A, Stegemann C, Nissum M, Zeng L, Brunet M, Mayr M. Proteomic analysis of the secretome of human umbilical vein endothelial cells using a combination of free-flow electrophoresis and nanoflow LC-MS/MS. Proteomics, 2009; 9(21): 4991-4996.

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