Research in the Smith group is directed towards the development of powerful new technologies to drive biological research. The work is multi-faceted and highly interdisciplinary and collaborative in nature. Two major areas of interest are surface chemistries, particularly those related to the development and use of various sorts of biomolecular arrays, and mass spectrometry. An emerging area of interest is in the field of synthetic biology, which has as its goal the development of a sufficient understanding and control of the fundamental processes of life to be able eventually to create self-replicating and sustainable entities.
The advent of high-density DNA arrays in the early ‘90s demonstrated the power of the array concept for genome-wide analyses of biological systems. Through a collaboration with Professor Franco Cerrina in the Department of Electrical Engineering and Computer Science, our group has acquired a Maskless Array Synthesizer, which allows any high-density DNA array of interest (up to 786,000 individual DNA features) to be designed and fabricated overnight. Recently we have developed a novel lamellar substrate for such DNA array fabrication, consisting of a thin layer of amorphous carbon deposited on a gold thin film over glass. The gold thin film supports the generation of surface plasmons, which are collective oscillations of electron density within the gold layer, and thereby allows the technique of surface plasmon resonance (SPR) imaging to be utilized for label-free detection on these carbon surfaces. Carbon attachment chemistry that we have developed over the last several years, permits biomolecular arrays of unprecedented chemical stability to be made on these surfaces. We are actively exploring applications of these new materials for the parallel analysis of DNA:DNA, DNA:RNA, DNA:protein, and protein:small molecule interactions. Together with Professor Helen Blackwell of the Department of Chemistry, we are developing and applying such tools to study quorum-sensing pathways in gram-negative pathogens such as Pseudomonas aeruginosa.
Biology has entered a new era with the recent sequencing of the human and many other genomes. With thousands of genome sequences now readily accessible in databases, research paradigms have fundamentally changed. One of the best examples of this new approach is in the field of proteomics, where tandem mass spectrometric analyses of complex protein mixtures depend upon whole genome database search algorithms to identify proteins. This approach exploits synergies between genome analysis, bioinformatics, and rapidly evolving instrumentation and chemistries for mass spectrometry.
As powerful as this technology has become, we believe that the field of biological mass spectrometry is still in its infancy. Mass spectrometry as it currently exists is a relatively inefficient process, in which often only one out of 107 to 1010 molecules in a sample being analyzed actually give rise to a detection event. This is because of ion losses that occur throughout a mass spectrometry system, in the ion source, the mass analyzer, and at the detector. Although the resolution for low m/z species can be extremely high, at larger m/z values corresponding to large biomolecules and biomolecular complexes in low charge states, both resolution and detection efficiency are extremely poor. Our group is interested in addressing the fundamental issues that limit biological mass spectrometry. We have active projects to improve ionization processes, reduce ion suppression and matrix effects, develop a new generation of highly sensitive ion detectors, and develop approaches for the determination of accurate masses of proteins in complex mixtures. We are also actively engaged in proteomics collaborations encompassing a variety of areas, such as human embryonic stem cells (Prof. Thomson, Genome Center of Wisconsin), angiogenesis (Profs. Olivier and Greene, Medical College of WI), the proteasome complex in Arabidopsis (Prof. Vierstra, Dept. of Genetics, UW-Madison), vocal chord function (Prof. Welham, Dept. of Surgery, UW-Madison), breast cancer (Prof. Gould, UW Comprehensive Cancer Center), and several others. These real-world projects keep us at the cutting edge of the rapidly evolving world of biological mass spectrometry, while helping to provide important information essential to understanding these fascinating and important biological systems.
Tools for understanding gene regulation
The successful completion of sequencing the human and other genomes has ushered in a new era in biological research. A strong focus now is identifying regulatory mechanisms that turn genes on or off, and then understanding how such gene regulation is altered by critical biological processes, diseases, or environmental factors such as drugs. We have recently established the Wisconsin Center of Excellence in Genomic Sciences, where together with Profs. Coon, Gasch and Thomson at UW Madison and Prof. Olivier at the Medical College of Wisconsin, we are developing novel technologies to identify the proteins that bind to particular DNA regions and control gene expression. Briefly the technology involves (1) chemical cross-linking of proteins to DNA, (2) fragmentation of the chromatin (long strands of DNA wrapped around proteins), (3) capture of these fragments onto surfaces in a DNA-sequence-specific manner, and (4) mass spectrometry to identify and quantify the proteins.
A new area of interest in our group is synthetic biology. There are numerous cell-free systems that can be used to express proteins from a gene of interest. In the presence of liposomes, these systems have been used to express fully functional membrane proteins that are inserted into the lipid membrane of the liposome. We are interested in using this technology to express functional, multi-component systems. One project involves combining the input signal from native signal transduction pathways with a novel output in order to detect sub-threshold levels of molecules of interest. Another project aims to create a novel, in vitro assay to detect differences in cytochrome P450 xenobiotic metabolism. In the long term, it may eventually be possible to combine this technology with artificial genomes to create self-replicating entities, the ultimate goal of biological engineering.