Advances in nano-liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) have enabled the analysis of increasingly high complexity and low abundance biological molecules, including extensively co- and post-translationally modified proteins. Receptor tyrosine kinases (RTKs) are low abundance cell surface receptors for cytokines, hormones, and growth factors. Dysregulation of RTK signaling plays a critical role in many diseases, including cancer, and RTKs are important therapeutic targets. Most RTKs are highly N-glycosylated. Changes in glycosylation can impact receptor trafficking and signaling, with important implications for cancer development, progression, and treatment. We aimed to apply nanoLC-MS/MS to study the N-glycosylation in two RTKs, vascular endothelial growth factor receptor-2 (VEGFR-2) and epidermal growth factor receptor (EGFR). Developing methods to study RTK glycosylation is a critical step toward understanding how N-glycosylation impacts RTK signaling. Tryptic digests of immunoprecipitated VEGFR-2 and EGFR were analyzed on a Q Exactive™ HF Hybrid Quadrupole-Orbitrap™ mass spectrometer (Thermo Scientific) with a nanoAcquity UPLC system (Waters) and a Triversa Nanomate (Advion, Ithaca, NY). A nanoACQUITY UPLC Symmetry C18 Trap Column (100 Å, 5 μm, 180 μm × 20 mm, Waters) was used for trapping, and an ACQUITY UPLC Peptide BEH C18 nanoACQUITY Column (130 Å, 1.7 μm, 150 μm × 100 mm, Waters) was used for chromatographic separation. The QE-HF MS was operated in data-dependent mode; the top 20 precursors were selected for fragmentation, with a stepped normalized collision energy (NCE) of 15% and 35%. Using nanoLC-MS/MS methods with a stepped collision energy, we have detected glycopeptides spanning the majority of the VEGFR-2 and EGFR N-glycosylation sites, and observe extensive N-glycan heterogeneity at many sites. The MS/MS spectra contain peptide b- and y-ion fragments that facilitate peptide identification, as well as carbohydrate fragments that give topological information regarding the carbohydrate modifications. Next, we used these methods to measure changes in site-specific glycosylation of VEGFR-2 and EGFR in response to biological perturbations and pharmacological intervention to demonstrate the measurement of biologically relevant, site-specific changes in RTK glycosylation. Application of these methods to the study of RTK signaling has high potential to elucidate how altered RTK glycosylation impacts cell signaling in cancer.

Exploring receptor tyrosine kinase N-glycosylation and signaling via nanoLC-MS/MS using higher-energy collisional dissociation and stepped collision energy

256th National Meeting (2018)

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