This proposal describes continuation of our research in developing highly sensitive and informative analytical methodologies beneficial for glycobiology research. Detailed structural information is now routinely required from minute quantities of complex glycoconjugates through an effective combination of microisolation, microcolumn separation and mass spectrometry. This includes oligosaccharide sequences, branching and linkages, all to be achieved at high sensitivity. The major methodological hurdles for submicrogram analysis of both N- and O-glycans were solved under the present grant. Efforts are now focused on the development of on-line microscale isolation of glycoprotein and enzymatic sequencing, labeling, separation and tandem MS analyses of N- and O-glycans derived from biological samples to facilitate better understanding of the biology underlying human diseases. Analytical advancements are being thought through the development of numerous methodologies including: (1) lectin microcolumns for on-line trapping of glycoproteins/glycopeptides, (2) endo-and exoglycosidase microreactors for faster and more efficient enzymatic characterization of N-glycans, (3) on-line per-O-methylation and coupling to LC/MSMS, (4) MS-based technology for the analysis of sialylated glycans, (5) derivatization of glycans to enhance separation and ionization, and (6) hydrophilic porous polymer monolith LC columns. The potential of the different proposed methodologies will be demonstrated in the areas of cancer research and alcohol abuse. The proposed methodologies will be employed to construct comprehensive glycomic/glycoproteomic maps for (a) biological samples (i.e., blood, tissue, breast duct lavage) obtained from cancer patients, and (b) brain and liver tissues obtained from rats with chronic alcohol exposure. The proposed analytical methodologies, which should result in pushing the sensitivity of current methodologies by several orders of magnitude, will prompt improved prevention, diagnosis, and treatment in the area of cancer research. They will also advance better understanding of alcohol abuse at the molecular level.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM024349-23
Application #
7087051
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Edmonds, Charles G
Project Start
1978-07-01
Project End
2009-06-30
Budget Start
2006-07-01
Budget End
2007-06-30
Support Year
23
Fiscal Year
2006
Total Cost
$278,633
Indirect Cost
Name
Indiana University Bloomington
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
006046700
City
Bloomington
State
IN
Country
United States
Zip Code
47401
Zou, Guozhang; Benktander, John D; Gizaw, Solomon T et al. (2017) Comprehensive Analytical Approach toward Glycomic Characterization and Profiling in Urinary Exosomes. Anal Chem 89:5364-5372
Snyder, Christa M; Alley Jr, William R; Campos, Margit I et al. (2016) Complementary Glycomic Analyses of Sera Derived from Colorectal Cancer Patients by MALDI-TOF-MS and Microchip Electrophoresis. Anal Chem 88:9597-9605
Mitra, Indranil; Snyder, Christa M; Zhou, Xiaomei et al. (2016) Structural Characterization of Serum N-Glycans by Methylamidation, Fluorescent Labeling, and Analysis by Microchip Electrophoresis. Anal Chem 88:8965-71
Struwe, Weston B; Agravat, Sanjay; Aoki-Kinoshita, Kiyoko F et al. (2016) The minimum information required for a glycomics experiment (MIRAGE) project: sample preparation guidelines for reliable reporting of glycomics datasets. Glycobiology 26:907-910
Novotny, Milos V; Alley Jr, William R (2013) Recent trends in analytical and structural glycobiology. Curr Opin Chem Biol 17:832-40
Alley Jr, William R; Mann, Benjamin F; Hruska, Vlastimil et al. (2013) Isolation and purification of glycoconjugates from complex biological sources by recycling high-performance liquid chromatography. Anal Chem 85:10408-16
Mann, Benjamin F; Mann, Amanda K P; Skrabalak, Sara E et al. (2013) Sub 2-?m macroporous silica particles derivatized for enhanced lectin affinity enrichment of glycoproteins. Anal Chem 85:1905-12
Novotny, Milos V; Alley Jr, William R; Mann, Benjamin F (2013) Analytical glycobiology at high sensitivity: current approaches and directions. Glycoconj J 30:89-117
Alley Jr, William R; Mann, Benjamin F; Novotny, Milos V (2013) High-sensitivity analytical approaches for the structural characterization of glycoproteins. Chem Rev 113:2668-732
Alley Jr, William R; Novotny, Milos V (2013) Structural glycomic analyses at high sensitivity: a decade of progress. Annu Rev Anal Chem (Palo Alto Calif) 6:237-65

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