Future advances in biochemical research and biomedical sciences are greatly dependent upon analytical separation methods. Among the recent important developments in separation science, microcolumn liquid chromatography and capillary electrophoresis are uniquely suited to deal wit exceedingly complex biological mixtures ad the challenges of extremely sensitive detection and structural characterization. The present proposal is targeted toward significant improvements in the general capabilities of these separation methods for structural studies and analysis of the most important biological macromolecules, proteins and ribonucleic acids. While microcolumns will be employed in the separation and microisolation of biomolecules, their coupling with laser- based fluorescence detectors ensures extremely high detection sensitivity for degradation products of the biomacromolecules, such as amino acids, peptides, ad nucleotides., The proposed developments will enable analysis in the range of 0-15 - 10-18 gram quantities. Consequently, the important techniques of sequencing and peptide mapping will make it possible to characterize roughly 10,000-times smaller amounts of biomolecules than is currently feasible. These advances will enable work with extremely small volumes of biological fluids, approaching detection at the level of single cells. Whenever feasible, components of separated mixtures will be identified by mass spectrometry, which couples conveniently with the miniaturized columns. Extremely high sensitivities obtained through laser-based detection are primarily due to unique synthetic designs of fluorogenic reagents in this laboratory. These powerful new bioanalytical techniques will be applied to tow problems of biomedical significance: (a) studies of interactions between model proteins and aldehydic neurotoxins and (b) characterization of the polypeptide carriers of reproduction-related pheromones in rodents.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Indiana University Bloomington
Schools of Arts and Sciences
United States
Zip Code
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
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
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

Showing the most recent 10 out of 111 publications