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.