Recently, researchers examined the link between glycosylation and endothelial protein secretion in the secretome of endothelial cells using a well-known secretagogue and three variations of mass spectrometry. The implications of this study may be far-reaching since the protein and/or glycoprotein-mediated changes in endothelial cells relate to various cardiovascular anomalies and dysfunctions, comprising the highest number of deaths and disabilities worldwide.
For the experiment, PMA was used to stimulate serum-starved human umbilical vein endothelial cells (HUVECs) coupled with glycoprotein/glycopeptide enrichment strategies. The glycopeptides were then analyzed in triplicate using three techniques: detection of 18O asparagine deamidation after digestion with PNGase F in H218O, alternating HCD-ETD, and HCD product-dependent ETD on an LTQ Orbitrap Elite mass spectrometer (Thermo Scientific).
Yin et al.1 identified 1,252 proteins, including 253 extracellular or plasma membrane proteins linked to the following cellular activities: cell adhesion, blood coagulation, hemostasis, signaling transduction, and protein transportation. This represents 20 percent of the total identified proteins. A total of 166 of these proteins were known to be glycosylated. The researchers then used the three mass spectrometry techniques listed above to quantify the glycopeptides and sites of glycosylation within the same sample.
From 110 identified spectra, HCD-alt-ETD revealed 20 known glycosylation sites, 32 potential sites, and 14 novel sites. This method primarily detected precursors with higher intensities, higher charges, and smaller m/z. This method was also solely responsible for the identification of several large glycopeptides and almost double the number of complex/hybrid glycans. From 209 identified spectra, HCD-pd-ETD revealed 28 known glycosylation sites, 25 potential sites, and 16 novel sites. This method more readily identified low abundant glycopeptides. Interestingly, the overlap between the two methods was limited in terms of identified glycopeptides but better for proteins in general.
The researchers verified glycosylation status using a Q Exactive mass spectrometer (Thermo Scientific) both before and after glycoprotein enrichment. A comparison of the glycoprotein-enriched portion, the input, and flow-through revealed that the majority of the glycoproteins exhibited a higher spectral count in the glycoprotein-enriched fraction compared with the input and/or flow-through. Nonglycosylated proteins, on the other hand, demonstrated higher abundance in the flow-through.
Among the benefits of this study, Yin et al.1 were able to minimize cell death, a common challenge in secretome analysis, and detected a larger number of proteins than other proteomics-based endothelial cell studies have accomplished thus far. These proteins include endothelial surface markers, secreted growth factors, coreceptors, proteases, and inflammatory mediators. Furthermore, the utilization of three complementary mass spectrometry methods offers an experimental model that features several advantages in terms of not only secretome analysis in particular but also increased confidence in accurate results and potential reproducibility in general.
Yin, X., et al. (2013) ‘Glycoproteomic analysis of the secretome of human endothelial cells‘, Molecular and Cellular Proteomics, published online January 23, 2013. doi: /10.1074/mcp.M112.024018