Proteins and other large biopolymers have tremendous clinical, biological and even environmental significance, yet it can be a difficult task to separate them in complex mixtures and to measure trace quantities present in such mixtures. Dr. Colyer's research program addresses these difficulties by developing new bioanalytical separation methods, with a particular emphasis on the utility of capillary electrophoresis with laser-induced fluorescence detection. The determination of phycobiliproteins and investigations into the mechanism of noncovalent binding of nonfluorescent proteins with visible and near-infrared organic dyes are examples of current projects in the Colyer lab.
Phycobiliproteins are water soluble, highly fluorescent proteins produced in nature by cyanobacteria (blue-green algae) and red algae. These proteins can absorb light energy across a region that is not efficiently absorbed by chlorophyll a, and so they serve to enhance the light-harvesting capabilities of their host seawater organisms. To facilitate phycobiliprotein determinations, we are developing high-speed and high-efficiency extraction methods, followed by high-sensitivity CE-LIF separation methods for not only the proteins themselves, but also for their "bilin" pigments and the intact microorganisms that create these proteins. Our studies will ultimately provide a routine analytical tool for quantifying the diagnostic phycobiliproteins in seawater, allowing for better measurements of cyanobacterial biomass and distributions, thus improving estimates of ocean primary production, and the role that oceans play in the global carbon cycle.
Noncovalent, near-infrared labels as facilitators of protein determination:
By coming to understand the nature of noncovalent interactions between various dye and protein molecules, it will be possible to develop efficient, high sensitivity analytical methods with applicability to the separation and quantitation of nonfluorescent proteins in complex mixtures. Noncovalent labeling strategies, coupled with CE and laser-induced fluorescence (LIF), are designed to overcome the disadvantages normally associated with protein derivatization, such as increased sample preparation and handling, restrictive solution pH and temperature conditions, and decreased separation efficiency due to the formation and partial resolution of multiple, differently-labeled species. To this end, appropriate buffer conditions are found to render visible and near-infrared dyes (especially squarylium dyes and boronic-acid based probes) non-fluorescent until noncovalently bound to a mixture of proteins, at which point a significant enhancement in the fluorescence of the bound dye will allow for detection of the proteins by CE-LIF. Our applied CE studies are accompanied by the investigation of some underlying physical phenomena that affect separation and on-column reaction performance. For example, the reaction kinetics of protein-dye interactions, the stability of protein-dye complexes, their stoichiometries and affinities, and their mobilities relative to those of unlabelled proteins, are important to the field of CE and to many other disciplines, too. As such, we design experiments to measure these parameters so that their impact can be quantitatively assessed.
Graduate and undergraduate students in our research group are exposed to many aspects of chemical research. Projects in our group allow students to develop a wide variety of skills beyond separation method development. These skills include basic biochemical sample handling, derivatization chemistry, biomolecule conjugation, laser optics, spectrophotometric methods, electronics, data acquisition, software programming, and statistical analysis. Students are encouraged to present their research work at regional, national, and international conferences, and are given opportunities to collaborate with researchers at other institutions.
S.C. Fordahl, J.G. Anderson, P.T. Cooney, T.L. Weaver, C.L. Colyer, and K.M. Erikson, Chronic manganese exposure inhibits the clearance of extracellular GABA and influences taurine homeostatis in the striatum of developing rats. Neurotoxicology. 31: 639-646 (2010).
X. Lin, A.R. Gerardi, Z.S. Breitbach, D.W. Armstrong, and C.L. Colyer, CE-ESI-MS Analysis of Singly Charged Inorganic and Organic Anions Using a Dicationic Reagent as a Complexing Agent. Electrophoresis. 30: 3918-3925, 2009.
X. Lin, J.P. Landers, and C.L. Colyer, Electrophoresis in Microfabricated Devices. In: J. Cazes, Editor: Dekker Encyclopedia of Chromatography (3rd Edition). Print ISBN 9781420084597, Taylor & Francis, Inc., NY. Oct. 15, 2009.
J.G. Anderson, S.C. Fordahl, P.T. Cooney, T.L. Weaver, and C.L. Colyer, and K. Erikson, Extracellular Norepinephrine, Norepinephrine Receptor and Transporter Protein and mRNA Levels Are Differentially Altered in the Developing Rat Brain Due to Dietary Iron Deficiency and Manganese Exposure. Brain Research. 1281: 1-14, 2009.
A.R. Gerardi, J.L. Lubbeck, and C.L. Colyer, Dimethylditetradecylammonium bromide (2C14DAB) as a self-assembled surfactant coating for detection of protein-dye complexes by CE-LIF. Journal of Solid State Electrochemistry. 13: 633-638, 2009.
J.G. Anderson, S.C. Fordahl, P.T. Cooney, T.L. Weaver, C.L. Colyer, and K.M. Erikson, Manganese exposure alters extracellular GABA, GABA receptor and transporter protein and mRNA levels in the developing rat brain. Neurotoxicology 29: 1044-1053, 2008.
K.D. Chichester, M. Sebastian, J.W. Ammerman, and C.L. Colyer, Enzymatic assay of marine bacterial phosphatases by capillary electrophoresis with laser induced fluorescence detection. Electrophoresis 29: 3810-3816, 2008.
A.L. Sloat, M.G. Roper, X. Lin, J.P. Ferrance, J.P. Landers, and C.L. Colyer, Protein determination by microchip capillary electrophoresis using an asymmetric squarylium dye: Noncovalent labeling and nonequilibrium measurement of association constants. Electrophoresis 29: 3446-3455, 2008.
X. Lin and C.L. Colyer, Chromatographic and electrophoretic methods for the determination of binding constants for dye-protein complexes. Journal of Liquid Chromatography & Related Technologies 31: 1620-1640, 2008.
K.D. Chichester, D.B. Silcott, and C.L. Colyer, Analysis of Bg spores by capillary electrophoresis. Electrophoresis 29:641-651, 2008.