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Research Description:
Armed with sequence information of the human and mouse genomes, a major aim of biological science is toward unraveling the underlying molecular events that lead to cellular function/dysfunction in disease with the goal of discovering better diagnostic markers and therapeutic targets. Proteomics aims to facilitate this process by applying newly developed methods and advanced analytical tools for the investigation of the protein complement and its repertoire of post-translational modifications en masse. Our efforts are, therefore, focused on development of new technologies that bridge the fields of chemistry and biology toward their application for characterization of proteomic changes associated with pathophysiology, ascribing to the philosophy that effective biomedical investigation mandates creative multidisciplinary approaches. In practice, we utilize advanced mass spectrometry (MS) tools, molecular biology, and bioinformatics to conduct molecular investigations of human disease. In practiced our research has three major focus areas that include: 1) global profiling of protein and metabolite abundance changes, 2) global and targeted characterization of protein post-translational modifications, and 3) high-throughput assay development based on the use of selected reaction monitoring MS. Quantitative Proteomics A major goal today in biomedical research is to understand the nature of the molecular events underlying pathophysiology with the goal of discovering novel biomarkers for early disease detection, for monitoring the response to therapy and, in the best case scenario, to predict the clinical outcome. These biomarkers can be categorized according to their clinical applications. Diagnostic markers are used to initially define the histopathological classification and stage of the disease, while prognostic markers can help forecast the development of disease and the prospect of recovery. Based upon the peculiarities of individual cases, the predictive markers can be applied to choose different therapeutic modalities. A biomarker could include patterns of single nucleotide polymorphisms (SNPs) or DNA methylation or changes in mRNA, protein, or metabolite abundances providing these patterns can be shown to correlate with the characteristics of the disease. It has been demonstrated, however, that there is often no predictive correlation between mRNA abundances and the quantity of the corresponding functional protein present within a cell. Since proteins represent the preponderance of the biologically active molecules responsible for most cellular functions, it is likely that direct measurement of protein expression can more accurately indicate cellular dysfunction underlying the development of disease. The predominant method of measuring changes in protein expression utilizes two-dimensional polyacrylamide gel electrophoresis technologies where protein spot intensities are compared between gels on which two separate protein extracts have been resolved. In recent years, however, higher throughput methods, such as stable isotope labeling by amino acids in cell culture (SILAC), have been developed to measure changes in relative protein abundance levels by differentially labeling the samples with stable isotopes. In a SILAC experiment (Fig. 1), two different cell populations are separately grown in normal culture medium and that supplemented with either a single or multiple amino acids that possess an amino acid labeled with ‘heavy’ stable isotope atoms (13C, 2H, 15N, or 18O). The result of growth of cells in medium supplemented with ‘heavy’ isotope amino acids results in their incorporation into proteins producing proteins identical to those from cells grown in normal medium in all respects with the exception that they possess a greater mass according to the number of ‘heavy’ amino acids in the sequence. These strategies rely on multidimensional separations of complex proteome mixtures and on the ability of mass spectrometry to resolve the isotopically labeled samples to provide a measure of the abundances of two distinctly labeled samples. A major focus of our research, therefore, is toward applying quantitative proteomic methodologies for disease mechanism investigations.
Figure 1: An illustration of the SILAC workflow for quantifying relative changes in protein abundance from two cell populations in culture. Post-Translational Modification Analyses Most of the initial efforts in global proteomics have been focused on methods to effectively identify a large number of proteins in a rapid fashion. This type of simple identification, however, does not adequately describe a protein, in terms of either structure or function. Descriptors such as a protein’s expression level, its tertiary structure, its location within the cell, and its interactions with other biomolecules are critical to the function of a protein. One of the key factors that affect its function is the presence of post-translation modifications (PTMs). One of the most important PTMs used to modulate protein activity and propagate signals within cellular pathways and networks is phosphorylation. Cellular processes ranging from cell cycle progression, differentiation, development, peptide hormone response, and adaptation are all regulated by protein phosphorylation. While methods to effectively identify and determine relative protein abundances have been developed, the delineation of the function of a protein based solely from abundance changes still provides only a limited view of the proteome since numerous vital activities of proteins are modulated by phosphorylation. Many techniques have been developed over the past few decades to determine if a protein is phosphorylated. The predominant method has been the use of affinity reagents such as anti-phosphoamino acid-specific monoclonal antibodies. While these affinity-based detection methods can determine if a protein is phosphorylated they may not necessarily identify the specific site of modification. Knowledge of the sites of modification is important since an identical modification at a different site within the same protein can have widely a different effect on the protein’s activity. In addition, several different enzymes may modify a single protein, each providing an indication into which cell pathway may be active. Mass spectrometry provides the best available technology for the site-specific identification of post-translational modifications. The attributes of MS that are used to measured masses as well as obtain sequence information of peptides are directly applicable to the site-specific identification of modifications. 
Figure 2. Matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrum of a phosphopeptide. The loss of 98 Da is readily apparent in this tandem mass spectrum of a phophoseryl-containing peptide (FQS*EEQQQTEDELQDK) from β-casein. Hypothesis Driven or Candidate-based Protein Biomarker Assay Development using Selected Reaction Monitoring Mass Spectrometry Accurate quantification of proteins and peptides is a challenging problem because of the complexity and extreme dynamic range presented in biological samples. The widely adopted survey approach to proteomics, in which an attempt is made to detect all components, has proven to be limited in sensitivity towards low abundance proteins and typically provides limited quantitative accuracy and precision. The alternative hypothesis-driven or candidate-based approach relies on specific assays optimized for quantitative detection of selected proteins that can provide significantly increased sensitivity (e.g. pg/ml range) and precision (CVs < 5-10%) where the cost is in restricting the discovery of potential novel proteins. In practice, a combination of these approaches (one or more survey approaches for de novo biomarker discovery, coupled with a candidate-based approach to biomarker validation in large sample sets) is likely to provide an effective staged pipeline for generation of valid biomarkers of disease, risk and therapeutic response. Candidate-based specific assays rely on the specificity of capture or detection methods to select a specific molecule as analyte. Capture reagents such as antibodies can provide extreme specificity (particularly when two different antibodies are used, as in a sandwich immunoassay), and form the basis of most existing clinical protein assays. Mass spectrometry provides an alternative assay approach, relying on the discriminating power of mass analyzers to select a specific analyte and on ion current measurements for quantitation. In the field of analytical chemistry, many small molecule analytes (e.g., drug metabolites, hormones, protein degradation products and pesticides) are routinely measured using this approach at high throughput with great precision (CVs <5%). Most such assays employ electrospray ionization followed by two stages of mass selection: a first stage (MS) selecting the mass of the intact analyte (the molecular ion) and, after fragmentation of the molecular ion by collision-induced dissociation with gas atoms, a second stage (MS/MS) selecting a specific fragment of the parent, collectively generating a “selected reaction monitoring” (SRM, plural MRM) assay. The two mass filters produce a very specific and sensitive response for the selected analyte, which can be used to detect and integrate a peak in a simple one-dimensional chromatographic separation of the sample. In principle, this MS-based approach can provide absolute structural specificity for the analyte, and, in combination with appropriate stable-isotope labeled internal standards (SIS), it can provide absolute quantitation of analyte concentration. These measurements have been multiplexed to provide 30 or more specific assays in one run. Such methods are slowly gaining acceptance in the clinical laboratory for the routine measurement of endogenous metabolites (e.g., in screening newborns for a panel of inborn errors of metabolism) and some drugs (e.g., immunosuppresants). Based on this rationale, a significant aspect of our research is focused on utilizing the novel findings forthcoming from biomarker investigations in the abundance-based and post-translational modification analysis programs described above, to develop high-throughput, targeted assays based on the use of SRM-MS.
Education:
B.S. (Biochemistry/Biophysics), Washington State University, Pullman, WA, 1994.
Ph.D. (Biochemistry), The Ohio State University, Columbus, OH, 1999.
Postdoctoral Fellow, Pacific Northwest National Laboratory, Richland, WA, 1999-2000.
Important Publications:
- Flint MS, BL Hood, M Sun, N Stewart, J Jones-Laughner and TP Conrads. Proteomic analysis of the murine liver in response to a combined exposure to psychological stress and 7,12-dimethylbenz(a)anthracene. J Proteome Res 9:509-520, 2010
- Hood BL, J Grahovac, MS Flint, M Sun, N Charro, D Becker, A Wells and TP Conrads. Proteomic analysis of laser capture microdissected melanoma cells from skin organ cultures. J Proteome Res, in press, 2010
- Flint MS, G Kim, BL Hood, NW Bateman, NA Stewart and TP Conrads. Stress hormones mediate drug resistance to paclitaxel in human breast cancer cells through a CDK-1-dependent pathway. Psychoneuroendocrinology 34:1533-1541, 2009
- Vanacore R, A Ham, M Voehler M Sanders, TP Conrads, T Veenstra, PE Dawson, BK Sharpless and BG Hudson. A sulfilimine bond identified in Collagen IV. Science 325:1230-1234, 2009
- Abbatiello SE, YX Pan, M Zhou, AS Wayne, TD Veenstra, SD Hunger, MS Kilberg, JR Eyler, NGJ Richards and TP Conrads. Mass spectrometric quantification of asparagine synthetase in circulating leukemia cells from acute lymphoblastic leukemia patients. J Proteomics 71:61-70, 2008
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