Epidermal growth factor receptor (EGFR) plays a key role in oral squamous cell carcinoma (OSCC). Despite significant advances in systemic cancer therapies, including radiation oncology and surgical techniques, many patients with these cancers still fail to be cured. In several reports, inhibition of the EGFR signaling pathways has been accomplished extracellularly with specific antibodies to block ligand binding or intracellularly with small molecule inhibitors. The extracellular domain of EGFR is known to be heavily glycosylated. N-glycosylation results in the formation of various noncovalent interactions between the glycans and the EGFR extracellular domain near the EGF binding site. To study the EGFR from an analytical perspective, mass spectrometry-based approaches are being used. For the development of the methods, three well-characterized glycoproteins (alpha-1-acid glycoprotein, transferrin, and fetuin) were used as references and treated under the same conditions. The protein backbones were analyzed through proteomic methods, mainly by digestion with the protease trypsin, which releases peptides and glycopeptides. The glycopeptides were characterized, as were the corresponding glycans which were released by treatment with the glycosidase PNGase F. The analyses of the resulting peptides, glycopeptides, and glycans, were conducted by MALDI-TOF MS and HILIC-C18-Q-TOF MS/MS. Selected fragmentation methods, e.g. CID, ETD, and ECD, were also used. Data obtained for the standard proteins and their released glycopeptides and glycans will be presented and will be compared to data from the glycopeptides and N-glycans of EGFR. Analysis of the glycosylation of the EGFR will serve as a good basis to understand the binding between proteoforms of EGFR and their ligands, which likely play a significant role in OSCC and HNC cancer development and response to therapy.

Analysis of epidermal growth factor receptor glycosylation by advanced mass spectrometric methods

256th National Meeting (2018)

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