Sequential N/O-glycosylation analysis of heavily glycosylated HIV-1 gp120 by combination of electron-transfer/higher-energy collisional dissociation and stepped collision energy/higher-energy collisional dissociation (EThcD-sceHCD)

The envelope (Env) glycoprotein on the surface of human immunodeficiency virus type 1 (HIV-1) which decorated with a dense array of glycans is a determinant for viral invasion and host immune response of HIV-1 and a major target for a preventive HIV-1 vaccine. Improved vaccine design requires an understanding of the detailed information about the glycan type on each glycosite. Here, we used our well-established sequential glycoproteomic workflow to characterize the N/O-glycosylation of HIV-1 gp120 at the level of native intact glycopeptides based on a stepped collision energy/higher-energy collisional dissociation (sceHCD) mass spectrometry (sceHCD-MS/MS), and a combined electron transfer/higher-energy collisional dissociation (EThcD) and sceHCD mass spectrometry (EThcD-sceHCD-MS/MS).