Increasing interest in production of protein-based pharmaceuticals (biotherapeutics) is accompanied by an increased need for verification of protein folding and correct disulfide bonding. Recombinant protein expression may produce aberrant disulfide bonds and could result in safety concerns or decreased efficacy. Thus, the thorough analysis of disulfide bonding is a necessity for protein therapeutics. The use of ETD facilitates this analysis because disulfide bonds are preferentially cleaved when subjected to ETD. Here, we make use of this well-characterized reaction to assign disulfide bonding networks by coupling the use of extracted ion chromatograms (XICs) of cysteine-containing peptides with ETD analysis to produce an efficient assignment approach for disulfide bonding. This method can be used to assign a disulfide pattern in a de novo fashion, to detect disulfide shuffling, and to provide information on heterogeneity, when more than one disulfide bonding pattern is present. The method was applied for assigning the disulfide-bonding network of a recombinant monomer of the HIV envelope protein gp120. It was found that one region of the protein, the V1/V2 loops, had significant heterogeneity in the disulfide bonds.

Electron transfer dissociation (ETD) is commonly used in fragmenting N-linked glycopeptides in their mass spectral analyses to complement collision induced dissociation (CID) experiments. The glycan remains intact through ETD, while the peptide backbone is cleaved, providing the sequence of amino acids for a glycopeptide. Nonetheless, data analysis is a major bottleneck to high throughput glycopeptide identification based on ETD data, due to the complexity and diversity of ETD mass spectra compared to CID counterparts. GlycoPep Detector (GPD) is a web-based tool to address this challenge. It filters out noise peaks that interfere with glycopeptide sequencing, correlates input glycopeptide compositions with the ETD spectra, and assigns a score for each candidate. By considering multiple ion series (c-, z- and y-ions) and scoring them separately, the software gives more weighting to the ion series that matches peaks of high intensity in the spectra. This feature enables the correct glycopeptide to receive a high score while keeping scores of incorrect compositions low. GPD has been utilized to interpret data collected on six model glycoproteins (RNase B, avidin, fetuin, asialofetuin, transferrin and AGP) as well as a clade C HIV envelope glycoprotein, C.97ZA012 gp140ΔCFI. In every assignment made by GPD, the correct glycopeptide composition earns a score that is about two-fold higher than other incorrect glycopeptide candidates (decoys). The software can be accessed at http://glycopro.chem.ku.edu/ZZKHome.php.

The viral envelope glycoprotein (Env) is the major target for antibody (Ab)-mediated vaccine development against the Human Immunodeficiency Virus type 1 (HIV-1). Although several recombinant Env antigens have been evaluated in clinical trials, only the surface glycoprotein, gp120, (from HIV-1 subtype B, MN, and subtype CRF_01AE, A244) used in the ALVAC prime-AIDSVAX gp120 boost RV144 Phase III HIV vaccine trial was shown to contribute to protective efficacy, although modest and short-lived. Hence, for clinical trials in southern Africa, a bivalent protein boost of HIV-1 subtype C gp120 antigens composed of two complementary gp120s, from the TV1.C (chronic) and 1086.C (transmitted founder) HIV-1 strains, was selected. Stable Chinese Hamster Cell (CHO) cell lines expressing these gp120s were generated, scalable purification methods were developed, and a detailed analytical analysis of the purified proteins was conducted that showed differences and complementarity in the antigenicity, glycan occupancy, and glycan content of the two gp120 molecules. Moreover, mass spectrometry revealed some disulfide heterogeneity in the expressed proteins, particularly in V1V2-C1 region and most prominently in the TV1 gp120 dimers. These dimers not only lacked binding to certain key CD4 binding site (CD4bs) and V1V2 epitope-directed ligands but also elicited reduced Ab responses directed to those epitopes, in contrast to monomeric gp120, following immunization of rabbits. Both monomeric and dimeric gp120s elicited similarly high titer Tier 1 neutralizing Abs as measured in standard virus neutralization assays. These results provide support for clinical evaluations of bivalent preparations of purified monomeric TV1.C and 1086.C gp120 proteins.

Studying protein O-glycosylation remains an analytical challenge. Different from N-linked glycans, the O-glycosylation site is not within a known consensus sequence. Additionally, O-glycans are heterogeneous with numerous potential modification sites. Electron transfer dissociation (ETD) is the method of choice in analyzing these glycopeptides since the glycan side chain is intact in ETD, and the glycosylation site can be localized on the basis of the c and z fragment ions. Nonetheless, new software is necessary for interpreting O-glycopeptide ETD spectra in order to expedite the analysis workflow. To address the urgent need, we studied the fragmentation of O-glycopeptides in ETD and found useful rules that facilitate their identification. By implementing the rules into an algorithm to score potential assignments against ETD-MS/MS data, we applied the method to glycopeptides generated from various O-glycosylated proteins including mucin, erythropoietin, fetuin and an HIV envelope protein, 1086.C gp120. The site-specific O-glycopeptide composition was correctly assigned in every case, proving the merits of our method in analyzing glycopeptide ETD data. The algorithm described herein can be easily incorporated into other automated glycomics tools.

Entry of HIV-1 into target cells requires binding of the viral envelope glycoprotein (Env) to cellular receptors and subsequent conformational changes that culminates in fusion of viral and target cell membranes. Recent structural information has revealed that these conformational transitions are regulated by three conserved but potentially flexible layers stacked between the receptor-binding domain (gp120) and the fusion arm (gp41) of Env. We hypothesized that artificial insertion of a covalent bond will ‘snap’ Env into a conformation that is less mobile and stably expose conserved sites. Therefore, we analyzed the interface between these gp120 layers (layers 1, 2 and 3) and identified residues that may form disulfide bonds when substituted with cysteines. We subsequently probed the structures of the resultant mutant gp120 proteins by assaying their binding to a variety of ligands using Surface Plasmon Resonance (SPR) assay. We found that a single disulfide bond strategically inserted between the highly conserved layers 1 and 2 (C65-C115) is able to ‘lock’ gp120 in a CD4 receptor bound conformation (in the absence of CD4), as indicated by the lower dissociation constant (Kd) for the CD4-induced (CD4i) epitope binding 17b antibody. When disulfide-stabilized monomeric (gp120) and trimeric (gp140) Envs were used to immunize rabbits, they were found to elicit a higher proportion of antibodies directed against both CD4i and CD4 binding site epitopes than the wild-type proteins. These results demonstrate that structure-guided stabilization of inter-layer interactions within HIV-1 Env can be used to expose conserved epitopes and potentially overcome the sequence diversity of these molecules.