RESEARCH AND COLLABORATION
Abundance-based proteomicsA 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 separated. In recent years, however, higher throughput methods to measure changes in relative protein levels by differentially labeling the samples with stable isotopes have been developed. 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 is in applying quantitative proteomic methodologies for disease mechanism investigations.
Protein Post-translational Modification Analysis
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 a protein’s function 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. While MS has primarily been used for identification of phosphorylated sites, I have an active interest in developing MS-based tools that are capable of providing a quantitative measure of the extent of modification.
Hypothesis Driven or Candidate-based Protein Biomarker Assay Development using Selected Reaction Monitoring Mass Spectrometry
Accurate quantitation 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 (into the 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. There is intense interest in miniaturizing sets of such assays in array formats, although significant problems remain in the production of suitable antibodies and in the simultaneous optimization of multiple assays in one fluid volume. 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) 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.
Integration of Technology Development and Basic Discovery Research for Mechanistic Investigations of Disease and Biomarker Discovery
Our ultimate goal is to create an integrated platform for conducting functional proteome analyses of disease. This platform should operate from the earliest stages of protein discovery to the final validation of therapeutic potential, allowing the biology (rather than technology) to dictate the selection of potential targets for further validation and investigation. This objective can be realized through the confluence of innovative proteomic technologies and focused basic discovery research. The technology offers unprecedented opportunities to identify proteins mechanistically linked to human disease. These proteins will be incorporated into a basic discovery research program, with the goal of developing specific reagents to test and validate their function (e.g. antibodies, RNAi probes, etc.). The phenotypic impact of perturbing these proteins (by chemical and/or genetic methods) will be determined through a combination of analytical chemistry and cell biology. Taken together, the output of this platform is an integrated workflow for conducting fundamental discoveries of human disease with a pipeline to translating these discoveries to the clinic.
On-going Scientific Collaborations
University of Pittsburgh
Dr. William L. Bigbee, Department of Pathology
Dr. Jill Siegfried, Department of Pharmacology
Dr. Robert Sobol, Jr., Department of Pharmacology
Dr. Jennifer Grandis, Surgical Oncology
Dr. Emanuela Taioli, Department of Cancer Epidemiology
Dr. Adam Brufsky, Magee-Womens Hospital
Dr. Kristin Zorn, Magee-Womens Hospital
Dr. Robert Edwards, Magee-Womens Hospital
Dr. Uddhav Kelavkar, Department of Urology
Dr. Ivan Rosas, Department of Medicine
Dr. Robert Branch, Division of Clinical Pharmacology
Dr. Ed Jackson, Division of Clinical Pharmacology
Dr. Peter Shaw, Children’s Hospital, Univerisity of Pittsburgh
Dr. Ken Foon, Division of Hematology/Oncology
Dr. Patrick Moore, Molecular Virology, UPCI
Dr. Bruce Freeman, Chair, Department of Pharmacology
Dr. Steven Shapiro, Chair, Department of Medicine
Nationally
Dr. Nigel G. J. Richards, Department of Chemistry, University of Florida, Gainesville, FL: Development of a quantitative assay for asparagine synthetase for patients with recurrent acute lymphoblastic leukemia.
Dr. George R. Beck, Division of Endocrinology, Metabolism and Lipids, Emory University School of Medicine, Atlanta, GA: Proteomic and metabolomic investigations toward understanding the mechanism of osteoblast development.
Dr. Gary Siuzdak: Department of Molecular Biology and the Center for Mass Spectrometry, The Scripps Research Institute, La Jolla, CA: Proteomic and metabolomic investigations of viral infection.
Dr. Alan Wayne, Clinical Director of the Pediatric Oncology Branch and a Senior Clinician at the Center for Cancer Research, National Cancer Institute, National Institutes of Health: analysis of asparagine synthetase levels in childhood ALL blasts.
Internationally
Dr. Aly Karsan (Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia) – Biomarker investigations in mouse models of Lewis lung carcinoma.
Dr. Deborah Penque (Responsible Head, Proteomics Laboratory, Centro de Genética Humana, Instituto Nacional de Saúde Dr Ricardo Jorge, Lisbon, Portugal) – Biomarker investigations in chronic obstructive pulmonary disease.
