Characterization of Glycosylation Proﬁles of HIV-1 Transmitted/Founder Envelopes by Mass Spectrometry (cid:1) †

The analysis of HIV-1 envelope carbohydrates is critical to understanding their roles in HIV-1 transmission as well as in binding of envelope to HIV-1 antibodies. However, direct analysis of protein glycosylation by glycopeptide-based mass mapping approaches involves structural simpliﬁcation of proteins with the use of a protease followed by an isolation and/or enrichment step before mass analysis. The successful completion of glycosylation analysis is still a major analytical challenge due to the complexity of samples, wide dynamic range of glycopeptide concentrations, and glycosylation heterogeneity. Here, we use a novel experimental workﬂow that includes an up-front complete or partial enzymatic deglycosylation step before trypsin digestion to characterize the glycosylation patterns and maximize the glycosylation coverage of two recombinant HIV-1 transmitted/founder envelope oligomers derived from clade B and C viruses isolated from acute infection and expressed in 293T cells. Our results show that both transmitted/founder Envs had similar degrees of glycosylation site occupancy as well as similar glycan proﬁles. Compared to 293T-derived recombinant Envs from viruses isolated from chronic HIV-1, transmitted/founder Envs displayed marked differences in their glycosylation site occupancies and in their amounts of complex glycans. Our analysis reveals that the glycosylation patterns of transmitted/founder Envs from two different clades (B and C) are more similar to each other than they are to the glycosylation patterns of chronic HIV-1 Envs derived from their own clades. (cid:3) 4 mg/ml) 1 (cid:5) l of PNGase F solution (500,000 U/ml) for a week at 37°C at pH 8.5. Endo H and Endo F3 deglycosylation experiments were performed by incubating (cid:7) 75 (cid:5) g of the Env (protein concentration of (cid:3) 4 mg/ml) with the enzyme solutions. For the Endo H deglycosylation experiment, samples were denatured with 2 M urea in 100 mM Tris buffer (pH 5.5), followed by the addition of 2 (cid:5) l of Endo H ( (cid:1) 5 U/ml). After thorough mixing, the reaction mixture was incubated for 48 h at 37°C. In the Endo F3 deglycosylation exper- iment, samples were deglycosylated by addition of 2 (cid:5) l of Endo F3 ( (cid:1) 5 U/ml) to a reaction mixture containing 75- (cid:5) g Env samples in 50 (cid:5) M NH 4 C 2 H 2 O 2 (pH 4.5). The reaction mixture was incubated for 2 weeks at 37°C. As a quality control experiment, the Endo F3 and Endo H studies were ﬁrst conducted on the HIV-1 Env B.JR-FL. The results from these experiments indicated that site occupancy analysis with Endo H and Endo F3 produced results identical to those that occurredwhen the site occupancy was determined by PNGase F. Desialylation with sialidase A was performed by adding 2 (cid:5) l of sialidase A ( (cid:1) 5 U/ml) to 75- (cid:5) g Env at pH 6.5, the mixture incubated overnight at 37°C. to tryptic digestion, the pH of the deglycosylated Env adjusted to 8.5 with 300 mM NH 4 OH. Deglycosylated Env samples digested as absorbance, was (cid:3) 4 mg/ml, and samples containing 75 (cid:5) g of the HIV-1 Envs were used for protein digestion. Proteins were denatured with 6 M urea in 100 mM Tris buffer (pH 8.5) containing 3 mM EDTA and were fully reduced using either 15 mM DTT or 5 mM TCEP at room temperature for 1 h. TCEP was used for samples treated with glycosidases due to its stability and efﬁciency at breaking disulﬁde bonds at low pH (25). Both TCEP- and DTT-reduced samples were alkylated with 20 mM IAM at room temperature for another hour in the dark. Excess IAM in both samples was quenched by adding DTT to a ﬁnal concentration of 25 mM, and the mixture was incubated for 20 min at room temperature. The reduced and alkylated Env samples were digested with trypsin (30:1 protein/enzyme ratio) at 37°C and incubated overnight, fol- lowed by a second trypsin addition under the same conditions. The resulting HIV-1 envelope protein digest was either directly analyzed by LC/ESI-FTICR MS or stored at (cid:6) 20°C until further analysis. To ensure reproducibility of the method, protein digestion was performed at least three times on different days with Env samples obtained from the same batch and analyzed with the same experimental procedure. Mass spectrometry. LC/ESI-FTICR MS experiments were performed using a hybrid linear ion-trap (LIT) Fourier transform (FT) ion cyclotron resonance mass spectrometer (LTQ-FT; ThermoScientiﬁc, San Jose, CA) directly coupled to the Dionex UltiMate capillary LC system (Sunnyvale, CA) equipped with a FAMOS well plate autosampler. Mobile phases utilized for the experiment consisted of solvent A (99.9% deionized H 2 O plus 0.1% formic acid) and solvent B (99.9% CH 3 CN plus 0.1% formic acid). Five microliters of the sample ( (cid:7) 7 (cid:5) M) was injected onto a C 18 PepMap 300 column (300- (cid:5) in the C1 region and N 241 VSTVQCTHGIKPVVSTQLLL N 262 GSLAEEEIIIR in the C2 region. These peptides contain one and two HexNAcs, and these species coelute with glycopeptides with complex-type glycans and peptide portion EVHNVWATHACVPTDPNPQEMVLA N 88 VTENFNMWK. In ad- dition, the nonglycosylated form of N 241 VSTVQCTHGIKPVVSTQLLL N 262 GSLAEEEIIIR was also observed. The peptide sequences were conﬁrmed from tandem-MS data. This is a demonstration of using endoglycosidases to identify site occupancy for glycopeptides.