Site-Specific Analysis of Bone Morphogenetic Protein Receptor Type 2 N -Glycosylation Reveals High Heterogeneity at Site Involved in Ligand Binding

High Resolution Image Download MS PowerPoint Slide The bone morphogenetic protein receptor type 2 (BMPR2) is expressed in multiple cell types, including endothelial cells, and mediates cell signaling via binding of transforming growth factor β (TGF-β) family ligands, contributing to balanced cell proliferation, apoptosis, and angiogenesis. BMPR2 N -glycosylation reportedly influences ligand binding. Here, we sought to develop a bottom-up nanoliquid chromatography-tandem mass spectrometry method to obtain evidence of site-specific N -glycosylation of human BMPR2. To optimize the observation of all three predicted N -glycosylation sites, three protease combinations were tested. Trypsin-Glu-C N -glycopeptides spanning all three putative BMPR2 N -glycosylation sites (N29, N84, and N100) were observed. Analyses of N -glycosylation site occupancy revealed that sites N29 and N84 are highly occupied, while a minor but detectable proportion of BMPR2 is not glycosylated at site N100. Glycopeptide analyses revealed N -glycan compositions consistent with hybrid and complex glycoforms with abundant fucosylation and sialylation. The nLC-MS/MS method established here will facilitate the study of BMPR2 N -glycosylation and its function in homeostasis and disease. Data are available via ProteomeXchange with identifier PXD066494.