Statement of Current and Future Research Interests
My research program is dedicated to the development and use of modern mass spectrometry techniques for the advancement of biomedicine. There are three primary focii within the Wood Group research program related to biomedicine: instrumentation development, proteomics, and synthetic polymer characterization.
Miniaturization of Electrospray (Nanospray)/Coupling to Capillary Separations and Its Application to Detection of Peptides/Proteins in Biological Fluids
In the last five years, the development of nanospray, in which very low sample volumes are required (and thus ideal for ionization of biomolecules from biological samples) has been of keen interest in mass spectrometry. However, current nanospray emitters using metallized coatings are not durable nor are they resistant to electrical discharge, making their lifetimes short-lived. Recently, we have developed novel nanospray emitters that use polyaniline as the conductive coating. These emitters are extremely durable, and are the first to have demonstrated reusability (J. Am. Soc. Mass Spectrom. 2000, 11, 659-663). Furthermore, these are resistant to destruction via electrical discharge due to the anti-static properties of polyaniline. Because of these properties, they are ideal for coupling to capillary separations techniques. Work is in progress with Prof. Luis Colon's group to couple his unique capillary columns with our unique nanospray emitters for separation and identification of biomolecules directly from biological fluids. The novelty of these emitters was discussed in the American Chemical Society journal Chemical Innovation as part of its "Heart Cut" series (Chemical Innovation, October 2000, p. 11).
Several collaborative ventures with a diverse set of investigators from around the globe are directed toward applying the nanospray methodology in the detection and quantification of peptides and proteins from biological fluids such as cerebrospinal fluid, amniotic fluid, and blood plasma.
As a consequence, there has been considerable interest from industry about using these nanospray emitters on their commercial electrospray mass spectrometers. This has lead to my founding of a start-up company called Nanogenesys, Inc. This corporation, located in Erie Co., NY, was founded to develop and commercial miniaturization techniques with application to biotechnology and nanotechnology. UB has submitted US and International Patents for this invention.
View an animation of the nanospray process.
Proteomics and Non-covalent Interactions
In the next five years, the thrust of our work will be applied research in biomedicine for: unraveling the nature and sites of protein heterogeneity; quality control analysis of recombinant enzymes; sequencing proteins of unknown structure; and identifying active sites of proteins (of known sequence) that have been selectively modified by affinity labels. We are particularly interested in utilizing such methodology in the identification of nucleotide binding sites for ATP-binding enzymes. Recently, we have proved that the enzyme glucokinase (GK), which is implicated in certain forms of maturity onset diabetes of the young (MODY), can be chemically modified and inactivated by the Arg-specific reagent phenylglyoxal. However, the locations of the implicated Arg residues are not known, nor is the amino acid sequence of GK from Bacillus stearothermophilus, the only commercially available strain of GK. Thus, a major initiative being addressed by my group is the amino acid sequencing and identification of phenylglyoxal-modified Arg residues in order to provide a more clear picture of the active site of GK, and perhaps lead to improved understanding of why natural genetic polymorphisms of GK lead to diabetes. Recently, we have begun collaborating with Professor John Wilson at Michigan State University to extend these studies to related mammalian brain hexokinase.
A second proteomics initiative is identification of paclitaxel (taxol) binding sites on tubulin. Paclitaxel is a potent anti-mitotic agent used in treatment of ovarian, breast, and cervical cancers which binds to tubulin protein to form microtubules, while inhibiting the dissociation of microtubules. Recently, using electrospray ionization mass spectrometry (ESI-MS), a technique which has emerged for detecting non-covalent molecular complexes, we have established the existence of non-covalent paclitaxel dimers in solution. A key experiment developed by our group can be used to establish whether or not a non-covalent complex was truly formed in the solution phase. In our experiment, hydrogen/deuterium (H/D) isotope exchange is allowed to occur by exposing paclitaxel to D2O, thereby allowing heteroatom-bound H atoms to exchange for D. If a molecular aggregate observed by ESI-MS were simply formed as an "artifact" of the ESI process, one would expect the degree of D incorporation in the multimer to be the sum of D incorporation in each monomer. However, if intramolecular bonds are formed between component monomers, then some of these heteroatom-bound H atoms will be protected from H/D exchange. Thus, the H/D exchange level in multimers formed in solution MUST necessarily be lower than the sum of H/D exchange levels in the individual monomers. Indeed, this is exactly what is observed in ESI-MS of paclitaxel. The paclitaxel monomer averages 0.87 + 0.02 exchanges, while the average for the paclitaxel dimer is 0.34 + 0.02 when paclitaxel is exposed to D2O for 1 h. The average number of exchanges for the paclitaxel dimer is less than half of the average number of exchanges for the paclitaxel monomer; this unequivocally establishes that some form of intramolecular bonding between paclitaxel molecules must be present in the solution because H/D exchange in the dimer is hindered relative to exchange in the monomer. Unliganded tubulin heterodimer, if present, must be at very low concentrations. Furthermore, prior studies have clearly established a 1:1 stoichiometry between paclitaxel binding to tubulin heterodimer in microtubules. The X-ray crystal structure of paclitaxel coupled with our recent ESI-MS findings suggest that paclitaxel dimers are formed readily, and may play an important role in binding tubulin. Our hypothesis is that in order to bind tubulin and form microtubules, paclitaxel must first form a non-covalent drug-drug heterodimer. Thus, based on the 1:1 stoichiometry of paclitaxel binding to tubulin protein heterodimers, our hypothesis would predict that the smallest intramolecular complex which can be formed between paclitaxel and tubulin is ab...P...P...ab (where ab is the non-covalent tubulin dimer, and P...P is the non-covalent paclitaxel dimer;... represents intramolecular non-covalent forces). An understanding of the paclitaxel-tubulin interaction would provide insight into the mode of action of paclitaxel, as well as provide fundamental information on the regulation of the microtubule system. This information may be essential in understanding the therapeutic properties of paclitaxel, and could lead to design of novel taxanes with improved efficacy and/or decreased toxic side effects. Thus, our specific aims are: First, to determine what the major form of paclitaxel (monomer, dimer, or possibly larger aggregate?) bound to tubulin is using ESI-MS, and to determine the stability dependence of the tubulin-paclitaxel complex as a function of tubulin concentration, paclitaxel concentration, solvent composition, pH, and Mg2+ concentration. Second, to determine the binding site of paclitaxel to tubulin using a proteolytic mapping approach and ESI-MS. Third, to determine if other bioactive taxane derivatives of paclitaxel, which show enhanced formation of microtubules relative to paclitaxel, also form non-covalent drug-drug complexes, and to determine if noncovalent complexes with tubulin can also be detected using ESI-MS.
We now have a two-dimensional electrophoresis system dedicated toward the purification of proteins for in-gel digestion for proteomics applications.
Synthetic Polymer Characterization
Of primary importance to our research group in a fundamentally unique polymer, polyaniline, which is capable of conducting electricity, which we use in coatings for nanospray emitters (see above). Currently, we are investigating the structural properties of polyaniline using mass spectrometry techniques (ESI-FTMS, LD-TOF, and LD-FTMS) in combination with surface science techniques (FTIR, XPS). In addition, a collaboration with Professor Joe Gardella (Dept. of Chemistry, SUNY-Buffalo) and Bausch & Lomb Healthcare involves using mass spectrometry to characterize substances (i.e., synthetic polymers) used in the fabrication of biomaterials. This includes using multiple mass spectrometry techniques: electrospray ionization Fourier transform mass spectrometry (ESI-FTMS) for characterization of extractables from biomaterials, time-of-flight secondary ion mass spectrometry (TOF-SIMS) for characterization of biomaterial surface components, and matrix-assisted laser desorption ionization (MALDI) with TOF and FTMS for the characterization of bulk mobile materials. The MALDI-FTMS capability was added in our laboratory recently thanks to the donation of a Finnigan FT/MS 2000 from IBM (Endicott, NY). Using these techniques, we can determine molecular weights of polymers in biomaterials directly; furthermore, the high mass accuracy advantage of FTMS allows us to characterize monomer repeat unit identity and end group composition from a single experiment.
Updated: Feb 1, 2005, Webmaster.