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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI094797-01A1
Application #
8249726
Study Section
Special Emphasis Panel (ZRG1-IMST-J (02))
Program Officer
Schultz, Alan M
Project Start
2012-08-01
Project End
2016-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
1
Fiscal Year
2012
Total Cost
$289,963
Indirect Cost
$89,963
Name
University of Kansas Lawrence
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
076248616
City
Lawrence
State
KS
Country
United States
Zip Code
66045