Gycosylation is one of the most important and most complex protein post-translational modifications. Studies have shown that the glycan moieties on glycoproteins play critical roles in structural modulation and function as specific binding ligands for endogenous receptors or exogenous agents in many biological processes such as protein trafficking, cell?cell signaling, and cellular adhesion. Alterations in glycomic profiles have been linked to various diseases, including cancer, neurodegenerative disorders, immunological diseases and cardiovascular problems. These implications urge researchers to develop innovative cutting-edge bioanalytical platforms for quantitative analysis of glycans to facilitate elucidation of the diverse biological roles of glycans and their roles in human diseases. Advances in mass spectrometry (MS)-based glycoproteomics and glycomics are increasingly enabling qualitative and quantitative approaches for site-specific structural analysis of protein glycosylation. However, quantitative analysis of native glycans remains extremely challenging due to high complexity and diversity of glycan structures, difficulty of synthesizing glycan standards, the relatively low response in MS detection, and the wide dynamic range of glycans in clinically relevant samples. The primary goal of this proposal is to develop several versatile mass defect-based multiplex tags for high- throughput quantification of glycans and glycopeptides in complex biological samples using high resolution mass spectrometry (MS) instrumentation and ion mobility (IM) MS coupled with multidimensional separation techniques. We propose the following specific aims:
Specific Aim 1 ? To develop and validate novel mass defect-based multiplex dimethyl pyrimidinyl ornithine (DiPyrO) tags for cost effective and high-throughput MS1-level relative quantification of N-glycans released from biological samples.
Specific Aim 2 ? To design and synthesize multiplex isobaric multiplex reagents for carbonyl containing compounds (SUGAR) tags for high-throughput MS2-level glycan characterization and relative quantitation.
Specific Aim 3 ? To develop and implement a novel capillary electrophoresis (CE)/porous graphite carbon (PGC)-LC-IM-MS platform for isomer-specific quantitative glycomics and glycoproteomics analysis, particularly ?2,3-/?2,6-sialylation ratio analysis, and construction of intact N-glycopetide, N-glycan, and deglycosylated peptide collision cross section (CCS) database facilitated by electron-transfer high energy collision dissociation (EThcD)-enabled highly confident identification. Collectively, our proposed experiments will develop novel enabling tools and will generate cost-effective and novel mass defect-based labeling reagents for robust, sensitive and accurate glycan analysis with enhanced quantitative performance and structural elucidation capabilities. The performance of these tags will be cross validated within the glycoscience community.

Public Health Relevance

Glycosylation is one of the most common posttranslational modification of proteins, with over 50% of proteins featuring covalently attached glycans. The structural diversity and wide dynamic range of various glycans serve to impart functional variance, with aberrant glycosylation implicated in numerous disease conditions. This project seeks to address technical challenges in detecting and quantifying diverse native glycan structures and intact glycopeptides by developing a suite of new methods and chemical reagents for high throughput quantifications of complex glycans. Both new labeling reagents permitting MS1-based and MS2-based quantitation and integrated instrumentation platforms will be developed to enable isomer-specific detection and high resolution MS quantification of glycans and glycopeptides. The successful completion of the proposed research will provide the scientific community with enabling, affordable and accessible tools to uncover the roles of carbohydrates in biological and disease pathways.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01CA231081-03
Application #
9982677
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Krueger, Karl E
Project Start
2018-08-03
Project End
2021-07-31
Budget Start
2020-08-01
Budget End
2021-07-31
Support Year
3
Fiscal Year
2020
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715