Vanderbilt University School of Medicine
Clinical Proteomic Technology Assessment for Cancer
Team Leader: Daniel Liebler, Ph.D.
Overall Project Goal:
The overall goal is to improve the reproducibility of shotgun and targeted quantitative mass spectrometry as well as to standardize tissue proteomic analysis. Clinical samples from patients with breast, lung and colorectal cancers will be used to define the application of shotgun proteome analysis platforms for the discovery and quantitation of cancer biomarkers.
Laboratory Studies:
In addition to performing inter-laboratory studies within the CPTC, this laboratory also utilizes unique approaches to study MS tissue proteomics. Highlights of these studies:
- Evaluation of five different shotgun proteomic methods and ways to improve throughput, reproducibility and standardization in both analytical methods and data analysis
- Comparison of depletion strategies for MS
- Perform targeted quantitative analysis using stable isotope dilution LC/MS/MS and reverse phase antibody array technology for candidate markers.
- Standardize platforms and data analysis tools to enable interlaboratory collaboration in biomarker discovery and analysis.
- Employ three different candidate discovery approaches: analysis of tumors, gene expression, and cancer biology
Specifically, the laboratory will systematically evaluate different shotgun proteomic analysis platforms to represent a transition from MudPIT to an efficient and reproducible platform. For example, the use of data dependent scanning will impose a standardized method for acquisition of MS/MS spectra directed at minimizing the semi-random sampling of medium and low abundance peptides by data dependent MS/MS. Additionally, candidate biomarkers will be obtained from three sources: 1) discovery proteomic analysis of tumors, precancerous lesions and biofluids 2) gene expression and profiling studies 3) cancer biology studies. Cancer specimens will be acquired through collaboration with three SPORE repositories. The use of targeted quantitative analysis by stable isotope dilution LC-MS/MS will be used to quantitate cancer-related proteins in biofluids from cancer tissues, literature reports and gene expression profiles. Establishment of systematic ways to standardize proteomic protocols among multiple laboratories will be achieved through a variety of ways including the use of abundant protein depletion methods with MALDI-MS spectral counting and RP gradient dependent LC-MS/MS. Evaluation of four affinity capture reagents in shotgun proteomics by MALDI- and ESI-MS-MS will also be evaluated. The resultant information/materials will be shared with the research community.
Team Expertise
Team Leader:
Daniel C. Liebler, Ph.D.
Professor of Biochemistry
Director of Proteomics
Mass Spectrometry Research Center
Director of the Jim Ayers Institute for Precancer Detection and Diagnosis
Vanderbilt-Ingram Cancer Center
Vanderbilt University School of Medicine
Dr. Daniel C. Liebler received a Ph.D. in Pharmacology from Vanderbilt while training in the laboratory of F. Peter Guengerich. He did postdoctoral training in the laboratory of Donald J. Reed at Oregon State University. From 1987-2003, Dr. Liebler served on the faculty of the Department of Pharmacology and Toxicology in the College of Pharmacy at the University of Arizona. From 1987 to 1998, his research program focused on the mechanisms of action of antioxidants in prevention of oxidative damage and cancer. His group defined antioxidant pathways and mechanisms of antioxidant action of vitamin E, carotenes and flavonoid antioxidants and established the use of mass spectrometry (MS)-based assays for products of antioxidant reactions as markers of antioxidant function. Since 1998, Dr. Liebler's program has focused on the application of MS-based proteomics approaches to address the problem of how reactive chemical intermediates cause damage to proteins.
The Liebler group developed two data analysis tools, SALSA and P-Mod, which enable discovery of modified protein forms through analysis of MS data, even when the chemical nature and sequence specificity of the modifications are not known beforehand. This approach has been integrated with affinity capture methods to evaluate electrophile-mediated protein damage on a proteomic scale and map over 1500 sites of chemical modification on over 800 human proteins. Dr. Liebler's laboratory has also extended these proteomic approaches to analyze the role of site-specific modifications in the function of signaling and sensor proteins that regulate responses to chemical toxicity and oxidative stress. The mapping of modifications has been augmented by application of stable isotope tagging methods to analyze the kinetics of protein modification reactions.
Dr. Liebler relocated to Vanderbilt in June 2003, where he has served as Director of Proteomics in the Mass Spectrometry Research Center. The Proteomics Laboratory has implemented methods and approaches developed in Dr. Liebler's laboratory and serves over 130 research groups at Vanderbilt. Most recently, Dr. Liebler has accepted Directorship of the Jim Ayers Institute for Precancer Detection and Diagnosis, which is dedicated to the discovery of proteomic markers for early cancer detection and for guiding therapy of established disease. Dr. Liebler's long-term research goals are to apply proteomics and related emerging technologies to identify markers of disease, therapeutic effect and toxicity and to characterize the roles of protein damage in chemical toxicity and disease.
Proteomic Technology Specialists
Daniel Liebler*, Richard Capriloi*, Gordon Mills, Amy-Joan Ham and Lisa Zimmerman
Clinical Cancer Researchers
Pierre Massion, Daniel Beauchamp, Nipun Merchant, Anaradha Chakravarthi and Adriana Gonzalez
Bioinformaticians
David Tabb*, Constantin Aliferis, Douglas Hardin, Scott Sobecki and Gregory Wernke
Biostatisticians
Yu Shyr*, Bonnie LaFleur and Dean Billheimer
Biologists
Robert Coffey*, David Carbone* and Carolos Arteaga*
* Denotes a member of the Internal Advisory Committee
Participating Institutions:
Vanderbilt University
M.D. Anderson Cancer Center
Other Essential Personnel:
Dale Cornett, Nancy Winters, Melinda Sanders, Jennifer Adams, Jonathon Brock, Kristin Carson, Shuo Chen, Harriet Davis, Josh Prashant, Alex Stantikov, Cynthia Sullivan, Prashant Joshi, Jonas Almeida, Yiling Lu, Corwin Joy, Natalie Wright, Katherine Stemke-Hale
About Vanderbilt:
Vanderbilt University was founded in 1873 through a $1 million gift from Cornelius Vanderbilt. Today, Vanderbilt University is a private research university for undergraduate and graduate/professional students. The university comprises ten schools, a distinguished medical center, a public policy center and The Freedom Forum First Amendment Center. Vanderbilt offers undergraduate programs in the liberal arts and sciences, engineering, music, education and human development as well as a full range of graduate and professional degrees. The combination of cutting edge research, liberal arts and a medical center creates an atmosphere where students and researchers solve complex questions affecting our health, culture and society.
