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.

Phycobiliprotein analysis:
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. These methodologies will be exploited in the development of a shipboard CE instrument with self-aligning laser detection system for near real-time monitoring of these important biomarkers. 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 near-infrared, cyanine dyes 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. Similarly, protein labeling methods involving various symmetric and unsymmetrical squarylium dyes are being developed, along with a comparison of the relative selectivity of binding for various dye types. Many of these dyes offer the added advantage of long wavelength absorbance and emission, and so are compatible with inexpensive and robust diode lasers as excitation sources. 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.

Recent Publications

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 (In press).

K.D. Chichester, D.B. Silcott, and C.L. Colyer, Analysis of Bg spores by capillary electrophoresis, Electrophoresis 29:641-651 (2008).

K.D. Chichester, D.B. Silcott, and C.L. Colyer, Analysis of Bg spores by capillary electrophoresis, Electrophoresis (in press).

W. Yan and C.L. Colyer, Investigating noncovalent squarylium dye—protein interactions by capillary electrophoresis – frontal analysis, J. Chromatogr. A 1135: 115-121 (2006).

W.Yan, A.L. Sloat, S. Yagi, H. Nakazumi, and C.L. Colyer, Protein labeling with red squarylium dyes for analysis by capillary electrophoresis with laser-induced fluorescence detection, Electrophoresis 27: 1347-1364 (2006).

H. Nakazumi, T. Ohta, H. Etoh, C.L. Colyer, Y. Hyodo and S. Yagi, Near-infrared luminscent bis-squaraine dyes linked by a thiophene or pyrene spacer for noncovalent protein labeling. Synthetic Metals 153: 33-36 (2005).

W.Yan and C.L. Colyer, Fluorimetric studies and noncovalent labeling of protein with the near-infrared dye HITCI for analysis by CE-LIF. Journal of Separation Science , 28: 1409-1415 (2005).

C.L. Colyer, C.S. Kinkade, P.J. Viskari, and J.P. Landers, Analysis of cyanobacterial pigments and proteins by electrophoretic and chromatographic methods. Anal. Bioanal. Chem. 382: 559-569 (2005).

C.L. Colyer, P.J. Viskari, and J.P. Landers, Electrophoresis in Microfabricated Devices. In: J. Cazes, Editor: Dekker Encyclopedia of Chromatography (2 nd Edition). Marcel Dekker, Inc., NY, pp. 530-542 (2005).