Glycoproteomic analysis of the secretome of human endothelial cells

Abstract

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. 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. In addition to 123 secreted proteins, the secretome was particularly rich in membrane proteins. 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. 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. 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. Two methods were compared: alternating HCD-ETD and HCD-product-dependent ETD. The former detected predominantly high-intensity, multiply charged glycopeptides, whereas the latter preferentially selected precursors with complex/hybrid glycans for fragmentation. Validation was performed by means of glycoprotein enrichment and analysis of the input, the flow-through, and the bound fraction. 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.

Fig. 1.

PMA treatment to stimulate EC secretion. Treatment of HUVECs with PMA, a commonly used secretagogue, resulted in a characteristic morphological change indicative of activation. A, immunofluorescence staining of vWF (green) and VE-cadherin (red) shows the exocytotic effect of PMA. B, PMA increased protein secretion in the conditioned media as confirmed via immunoblotting. C, relative to previous studies, more than twice as many secreted and plasma membrane proteins were identified. D, overlay of intracellular and secreted proteins by means of difference gel electrophoresis. In the left-hand panel, proteins in conditioned media of HUVECs are stained in green (+PMA) and red (−PMA), and cellular proteins are stained in blue. Results were reproduced with different biological replicates using reverse-labeling (right-hand panel: red, +PMA; green, −PMA). The protein corresponding to von Willebrand antigen 2 is highlighted with a box. Common proteins in the secretome and the cellular proteome are numbered in supplemental Fig. S2 and listed in supplemental Table S3.

Fig. 2.

Glycoproteomics. A, glycopeptide identification workflow. Comparison of direct and indirect glycopeptide detection using HCD-ETD and ¹⁸O-deamidation after PNGase F + H₂¹⁸O treatment, respectively: identified unique glycopeptides (B), unique glycosylation sites (C), and unique glycoproteins (D).

Fig. 3.

HCD-pd-ETD fragmentation. Full MS showing the different glycoforms of the same peptide sequence (A). Characteristic oxonium ion detected by HCD at m/z = 204.09 (B). This HexNAc signature triggered an ETD scan to identify the peptide sequence and confirm the glycosylation site (C).

Fig. 4.

Comparison of HCD-pd-ETD and HCD-alt-ETD. The two methods, HCD-pd-ETD (blue) and HCD-alt-ETD (red), displayed distinct distributions of the observed m/z, charge state, mass of identified peptides (M+H), and glycan mass, as well as the intensity of the precursor ions and the Byonics^TM score (all y-axes). The x-axes represent index numbers after proteins were sorted by their corresponding y-axis value from lower to higher (A). There was limited overlap in the identified glycopeptides (B). C, the HCD-pd-ETD method preferentially identified complex/hybrid glycans.

Fig. 5.

Glycoprotein enrichment for validation. A, spectral count of input, glycoprotein-enriched fraction (GP), and flow-through fraction (FT) from representative glycoproteins and non-glycoproteins. B, complementarity of the different methods (HCD-ETD, PNGase F + H₂¹⁸O treatment, and glycoprotein enrichment). Only 18 glycoproteins were consistently identified.

Fig. 6.

Sequence coverage for vWF. A, schematic illustration of vWF sequence. Coverage is highlighted in green, and potential glycosylation sites are shown in red. A large hexagon indicates a glycosylation site with a reference in the Uniprot database. By using the HCD-ETD (H) or PNGase F (P) method, we confirmed six N-glycosylation sites on vWF. B, ETD spectra of glycopeptides identified via HCD-ETD (N156, N211, N666, N1574). The following abbreviations are used: a, y, g, k = TMT modified Ala, Tyr, Gly, and Lys, respectively; c = carboxyamidomethylation of Cys; m = oxidation of Met.