The ultimate goal of this proposal is to develop broadly applicable methods to analyze glycosylation and disulfide bonding of glycoproteins and to apply those methods to the HIV Envelope protein (Env). This work would be accomplished by completing four specific aims: (1) Develop a new glycoproteomic method, where glycosylation site occupancy is quantified and glycoform heterogeneity is simultaneously characterized, all in one experiment. (2) Profile Env glycosylation site occupancy and glycopeptide heterogeneity to answer several important biological questions. (3) Develop an automated analysis protocol for identifying disulfide- linked peptides. (4) Characterize the disulfide bonding of Env to answer several important biological questions. The bulk of the work would be completed by combining expertise in biological sample handling, HPLC, and mass spectral (MS) data acquisition and analysis to develop the necessary tools. The utility of all the tools would be demonstrated by incorporating them to solve problems relevant to HIV vaccine development. Of particular note are studies in Aim 2, where the tools are used to correlate changes in glycan content with changes in vaccine efficacy. This work can broadly impact human health by providing fundamental insights into how post-translational modifications impact HIV vaccine candidates'efficacy. Likewise, methods for glycoprotein and disulfide analysis are enabling bioanalytical technologies that can be used in a variety of venues, from protein structure/function analyses to pharmaceutical development. The application work related to HIV proteins is done in collaboration with Dr. Barton F. Haynes at Duke University Medical Center and Dr. Bing Chen at Harvard University Medical School.
We aim to develop broadly applicable methods to analyze post-translational modifications of glycoproteins. Methods will be applied to various proteins, including HIV-Env based vaccine candidates. These analyses will be used to help understand how changes in the post-translational modifications impact HIV-1 vaccine performance.
|Go, Eden P; Hua, David; Desaire, Heather (2014) Glycosylation and disulfide bond analysis of transiently and stably expressed clade C HIV-1 gp140 trimers in 293T cells identifies disulfide heterogeneity present in both proteins and differences in O-linked glycosylation. J Proteome Res 13:4012-27|
|Zhu, Zhikai; Go, Eden P; Desaire, Heather (2014) Absolute quantitation of glycosylation site occupancy using isotopically labeled standards and LC-MS. J Am Soc Mass Spectrom 25:1012-7|
|Zhu, Zhikai; Su, Xiaomeng; Go, Eden P et al. (2014) New glycoproteomics software, GlycoPep Evaluator, generates decoy glycopeptides de novo and enables accurate false discovery rate analysis for small data sets. Anal Chem 86:9212-9|
|Desaire, Heather (2013) Glycopeptide analysis, recent developments and applications. Mol Cell Proteomics 12:893-901|
|Go, Eden P; Liao, Hua-Xin; Alam, S Munir et al. (2013) Characterization of host-cell line specific glycosylation profiles of early transmitted/founder HIV-1 gp120 envelope proteins. J Proteome Res 12:1223-34|
|Clark, Daniel F; Go, Eden P; Desaire, Heather (2013) Simple approach to assign disulfide connectivity using extracted ion chromatograms of electron transfer dissociation spectra. Anal Chem 85:1192-9|