HIV-gp120

Deciphering the glycosylation of viral envelope (Env) glycoprotein is critical for evaluating viral invasion and the host’s immune response, and developing preventive vaccines and antiviral drugs. However, the site-specific N- or O-glycosylation characterization of these highly glycosylated Env proteins is still very challenging, although glycoproteomics has made significant advances in sample preparation, mass spectrometry fragmentation technique, and data analysis software. Here, we presented a hybrid dissociation technique, EThcD-sceHCD, by combining electron transfer/higher-energy collisional dissociation (EThcD) and stepped collision energy/higher-energy collisional dissociation (sceHCD), and integrated EThcD-sceHCD into a sequential glycoproteomic workflow (N/O-GlycoHybrid). Using this workflow, we characterized the site-specific N/O-glycosylation of the human immunodeficiency virus type 1 (HIV-1) Env protein gp120. Comparing with commonly used fragmentation mode (e.g. HCD, sceHCD, EThcD), EThcD-sceHCD was more reliable at generating fragment ion types sufficient for the robust characterization of intact N/O-glycopeptide, and can achieve more in-depth intact glycopeptides identification. Finally, eighteen N-glycosites and five O-glycosites with attached glycans were assigned unambiguously from heavily glycosylated gp120 using EThcD-sceHCD-MS/MS. These results indicated that the N/O-GlycoHybrid can achieve better performance and simultaneous analysis of the N/O-glycosylation of a highly glycosylated protein containing many potential glycosites in one process. Knowledge of the glycosylation landscape of the Env glycoprotein will be useful for the development of vaccines and drugs targeted toward HIV-1.