The ultimate goal of this proposal is to develop a fully-automated approach for analysis of glycopeptides and to apply that approach to the analysis of several different HIV-Env glycoproteins. This work would be accomplished by completing three specific aims:
AIM 1 : Using MS data as the input, develop an algorithm that rapidly identifies glycopeptide compositions for singly glycosylated glycopeptides.
AIM 2 : Develop an algorithm that characterizes multiply glycosylated glycopeptides.
AIM 3 : Use the tools in AIMS 1 and 2 to screen for accessible and genetically conserved points on the HIV Env protein. The bulk of the work, Aims 1 and 2, would be completed by synergistically incorporating expertise in mass spectral (MS) data acquisition and analysis with expertise in software design/development. This work can broadly impact human health research because glycopeptide analysis is an enabling bioanalytical technology that can be used to screen for biomarkers of disease or disease state; it can be used to help verify the safety and consistency of glycoprotein-based pharmaceuticals; and it can be used to analyze glycopeptides on HIV Envelope proteins, a major target for HIV vaccine development. After completion of the automated glycosylation profiling system (Aims 1 and 2) this system would be used to identify the glycopeptide composition on a variety of HIV- Env vaccine candidates generated in collaboration with Dr. Barton F. Haynes at Duke University Medical Center. The method for this approach includes preparing tryptic digests of each of the glycoproteins, conducing HPLC-MS and LC-MS/MS analyses on the digested products, and interpreting the results using the developed glycopeptide analysis software. After completion of this work, the glycopeptide profiles of the Env proteins would provide information about epitope accessibility on Env. This is relevant to human health because identifying epitopes on the protein that are consistently exposed across HIV-1 clades is a first step in designing an HIV vaccine that mimics these epitopes and elicits broadly neutralizing antibodies to HIV-1.

Public Health Relevance

We aim to develop tools for the rapid analysis of glycopeptides. Glycopeptides are components of the HIV Env protein, and their analysis could be used to identify Achilles Heels (regions which are consistently exposed and thus vulnerable to antibodies) on the surface of the virus.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Instrumentation and Systems Development Study Section (ISD)
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Sheeley, Douglas
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University of Kansas Lawrence
Schools of Arts and Sciences
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Go, Eden P; Cupo, Albert; Ringe, Rajesh et al. (2016) Native Conformation and Canonical Disulfide Bond Formation Are Interlinked Properties of HIV-1 Env Glycoproteins. J Virol 90:2884-94
Go, Eden P; Herschhorn, Alon; Gu, Christopher et al. (2015) Comparative Analysis of the Glycosylation Profiles of Membrane-Anchored HIV-1 Envelope Glycoprotein Trimers and Soluble gp140. J Virol 89:8245-57
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; 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
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
Woodin, Carrie L; Maxon, Morgan; Desaire, Heather (2013) Software for automated interpretation of mass spectrometry data from glycans and glycopeptides. Analyst 138:2793-803
Zhu, Zhikai; Hua, David; Clark, Daniel F et al. (2013) GlycoPep Detector: a tool for assigning mass spectrometry data of N-linked glycopeptides on the basis of their electron transfer dissociation spectra. Anal Chem 85:5023-32
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
Desaire, Heather (2013) Glycopeptide analysis, recent developments and applications. Mol Cell Proteomics 12:893-901
Zhu, Zhikai; Su, Xiaomeng; Clark, Daniel F et al. (2013) Characterizing O-linked glycopeptides by electron transfer dissociation: fragmentation rules and applications in data analysis. Anal Chem 85:8403-11

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