National Center for Glycomics and Glycoproteomics

National Center for Glycomics and Glycoproteomics

Research Emphasis

The National Center for Glycomics and Glycoproteomics (NCGG) focuses on developing highly sensitive methodologies and new instrumental capabilities for glycomic and glycoproteomic analyses of complex biological systems. In collaboration with biological and biomedical investigators, conceptually important applications are being sought to emphasize the advantages of sensitive and accurate measurements as well as the structural information provided through bioinformatic tools in glycomics and glycoproteomics. The unique methodologies offered by the center include isotopically-coded glycan analysis by mass spectrometry (MS), high-energy collision-induced dissociation of permethylated glycans, photofragmentation MS, lectin fractionation of complex glycoprotein mixtures, microchip-based glycobiology, and tandem ion mobility spectrometry. The emphasis has been focused on interfacing microcolumn separations with the state-of-the-art MS in integrated analytical platforms to maximize analytical capabilities.

Current Research

In the area of glycomic measurements, the center has recently developed quantitative tools for conversion of various glycans to their fully methylated derivatives at nanoscale. This permits highly informative tandem MS identification and glycan profiling from complex glycoprotein mixtures and various biological materials. Using the isotopically-aided labeling, it has become possible to investigate aberrant glycosylation in diseases such as cancer and alcoholism. In the area of glycoproteomics, it has utilized unique preconcentration procedures (based on the lectin-glycoconjugate interactions) to profile complex glycoprotein/glycopeptide mixtures from minute quantities of tissue extracts and physiological fluids. The new analytical platforms are oriented toward finding disease biomarkers. Besides developing new chemical approaches to glycoconjugate analysis, our strong emphasis has been on designing new instruments and analytical capabilities. Current activities include sophisticated instrumentation of tandem ion-mobility spectrometry (IMS-IMS and IMS-IMS-IMS), photofragmentation MS and multiplexing MS techniques as well as microfluidic sample treatment and separations. The center's strong emphasis has been the optimum utilization of separation methodologies in conjunction with MS techniques and computer-aided bioinformatics. The newly developed tools are being tested through unique biomedical applications.

Resource Capabilities

Instruments

NCGG is equipped with numerous modern mass spectrometers, including an Applied Biosystems 4800 proteomic analyzer (MALDI/TOF/TOF MS), a Micromass Q-TOF mass spectrometer with nanoflow Z-electrospray, a Thermo-Finnigan LCQ Deca XP mass spectrometer, an XCT Ultra ion-trap mass spectrometer from Agilent Technologies, and LTQ FT equipped with ECD and IRMPD from ThermoElectron. All these mass spectrometers are configured with nano-LC systems (Agilent Technologies and Dionex). The NCGG facility has a Beckman-Coulter capillary electrophoresis MDQ system with a laser-induced fluorescence detector, an Agilent capillary electrophoresis system, several HPLC systems with UV, and fluorescence detectors, several GC systems with different detectors, and two high pH anion-exchange chromatographs with a pulsed-amperometric detection. The NCGG is also equipped for SDS-PAGE and 2-DE analysis, including, a Bio-Rad ProteomeWorks Spot Cutter, a Bio-Rad GS-800 Calibrated Densitometer, and a Bio-Rad Protean IEF Cell.

Special Features

Quantitative, isotopically-aided MS measurements of glycomic profiles (C-GlycoMAP). Computer-aided structural identification of complex carbohydrates. High-sensitivity glycoproteomic and general-proteomic investigations of complex biological samples through multidimensional capillary liquid chromatography, capillary electrochromatography, and electrophoresis, 2-D gel electrophoresis, microfluidic (microchip) separations and various forms of tandem mass spectrometry. Photofragmentation mass spectrometry. Tandem ion-mobility spectrometry/mass spectrometry. NCGG offers a one-week annual workshop involving lectures and a hands-on experience. Currently, two bioinformatic algorithms are web-hosted at www.ncgg.indiana.edu. One is focused on the automated interpretation of tandem MS data for glycans, while the other allows automated identification of protein glycosylation sites and their microheterogeneity from LC/MSMS analyses of protease-digested samples.

Publications

1. Merenbloom, S. I., Koeniger, S. L., Valentine, S. J., and et al., IMS-IMS/MS for separating peptide and protein fragment ions. Anaytical Chemistry 78:2802–2809, 2006.

2. Koeniger, S. L., Merenbloom, S. I., and Clemmer, D. E., Evidence for many resolvable structures within conformation types of electrosprayed ubiquitin ions. Journal of Physical Chemistry B 110:7017–7021, 2006.

3. Mechref, Y., Kang, P., and Novotny, M. V., Differentiating sialylated glycan structural isomers through MALDI/TOF/TOF tandem mass spectrometry. Rapid Communications in Mass Spectrometry 20:1381–1389, 2006.

4. Mechref, Y. and Novotny, M.V., Miniaturized separation techniques in glycomic investigations. Journal of Chromatography B 841:65–78, 2006.

5. Madera, M., Mechref, Y., Klouckova, I., and Novotny, M. V., Semiautomated high-sensitivity profiling of human blood serum glycoproteins through lectin preconcentration and multidimensional chromatography/tandem mass spectrometry. Journal of Proteomic Research 5:2348–2363, 2006.

6. Janecki, D. J., Beardsley, R. L., and Reilly J. P., Probing protein tertiary structure with amidination. Analytical Chemistry 77:7274–7281, 2005.

7. Tang, H., Mechref, Y., and Novotny, M. V., Automated interpretation of MS/MS spectra of oligosaccharides. Bioinformatics 21:i431–i439, 2005.

8. Kim, T.-Y., Thompson, M. S., and Reilly, J. P., 157nm photodissociation of peptides in a linear ion trap mass spectrometer. Rapid Communications Mass Spectrometry 19:1657–1665, 2005.

9. Devakumar, A., Thompson, M. S., and Reilly, J. P., Fragmentation of oligosaccharide ions with 157 nm vacuum ultraviolet light. Rapid Communications Mass Spectrometry 19:2313–2320, 2005.

10. Mechref, Y. and Novonty, M.V., Structural investigations of glycoconjugates at high sensitivity. Chemistry Review 102:321–370, 2002.