Broad Institute of MIT and Harvard
Measuring Cancer Biomarker Candidates by Targeted MS and Antibody Enrichment
Team Leader: Steven Carr, Ph.D.
Co-PIs: Amanda Paulovich, FHCRC and Leigh Anderson, Plasma Proteome Institute
Overall Project Goal:
The Broad Institute Team, led by principal investigator Steven Carr together with co-principal investigators Amanda Paulovich of the Fred Hutchinson Cancer Research Center and Leigh Anderson of the Plasma Proteome Institute, is developing new targeted, quantitative technologies base on mass spectromentry (MS) that aim to provide the critical bridge between discovery “omics” methods and creation of clinically deployable assays. These methods build on Multiple Reaction Monitoring (MRM) Mass Spectrometry (MS), an approach that has been used previously in clinical chemistry to measure small molecule drugs and metabolites, but now is being tailored for protein biomarkers. For protein biomarker measurements in plasma, they identify one or more “signature” peptides to serve as a quantitative surrogate for each protein to be monitored. Synthetic, stable-isotope labeled versions of each peptide are added into trypsin-digested plasma, and the ratios of labeled to unlabeled (sample-derived) peptides are measured by MRM-MS. These measurements determine the amount of each signature peptide, and hence, the amount of protein from which it is derived. The team has demonstrated that MRM-MS assays can be highly multiplexed enabling the quantitation of 10’s of proteins in a single analysis of patient plasma.
The team is developing a range of sample fractionation and enrichment methods to enable quantitation of proteins at the bottom of the nanogram/mL range or lower in plasma, a range where a large number of clinically useful markers reside. A major focus of the team is in developing and optimizing novel enrichment technology known as SISCAPA. In this approach, immobilized anti-peptide antibodies are used to capture and enrich the peptides of interest in a single step from blood. The captured peptides are subsequently released and measured by MRM-MS.
After optimization and characterization, the MRM-MS assays will be used to measure the levels of protein biomarker candidates in plasma from breast cancer patients and compared to the levels in healthy controls. Outcomes will demonstrate 1) the ability to make sensitive/specific assays quickly and inexpensively 2) that assays can be multiplexed, reducing the cost per analyte, and 3) the protocols and technology can be standardized and distributed.
Laboratory Studies:
In addition to performing inter-laboratory studies within the CPTC, this laboratory also utilizes unique approaches to study cancer. Highlights of these studies:
- Evaluation of target proteins and multiplexing through MRM
- Enrichment of lower abundant proteins using anti-peptide antibodies (SISCAPA)
- Establishment of standardized protocols and technologies for intra-laboratory studies and the scientific community.
- Evaluation of an antibody array platform for potential high throughput analysis.
Specifically, immunoaffinity enrichment (SISCAPA) coupled with MRM quantitation by MS to capture and detect signature peptides derived from ca. 200 cancer biomarker candidate proteins will be developed and evaluated. The use of SISCAPA will offer higher enrichment levels and greatly simplified sample handling relative to other fractionation/enrichment methods. Standardized protocols for these technologies will be developed among multiple laboratories using these platforms with the goal to apply the assays developed to measure breast cancer-relevant proteomic changes in human clinical specimens. Ultimately the assays will be used to measure protein biomarker candidate levels in ca. 200 select breast cancer and 200 control plasma samples at multiple sites. This will define the quantitative reproducibility and transportability of the methodology using standardized reagents and procedures.
Team Expertise
Team Leader:
Steven A. Carr, Ph.D.
Director of Proteomics
Broad Institute of MIT and Harvard
Dr. Carr leads the Proteomics and Biomarker Discovery efforts at the Broad Institute of MIT and Harvard. His current research involves development and application of novel, quantitative approaches for biomarker discovery and validation as well as phosphoproteomics to understand drug and disease mechanisms and provide biomarkers for human cancers, heart disease and metabolic disorders. The Carr lab collaborates with biologists and chemists to systematically identify proteins and their modifications whose abundance or form is modulated by disease or drug action, as well as to define physical and functional associations of protein constituents of regulatory and signaling pathways involved in health and disease. These studies involve analysis of complex biological specimens, such as tumor tissues or patient blood using protein chemistry and advanced separation methods together with state-of-the-art mass spectrometry.
For the last 25 years, Dr. Carr's research has focused on applying and developing proteomics methods in order to understand the mechanism of action of drug candidates and build an understanding of protein targets and their roles in disease. He is noted for developing methods for selective enrichment, detection and quantitation of posttranslational modifications such as phosphorylation and glycosylation in the proteome. While at GlaxoSmithKline (1984-2001) and Millennium Pharmaceuticals (2001-2004), he made significant contributions to the discovery and development of four marketed drugs (small and large molecule) and to numerous drugs in clinical trial. His groups at GSK and Millennium also produced one of the first examples of proteomics-derived biomarkers in use in the clinic and the first example of de-orphaning of a G-coupled protein receptor by mass spectrometry. He has over 130 peer-reviewed publications on development and use of proteomics and biological mass spectrometry. Dr. Carr is an Associate Editor of Molecular and Cellular Proteomics, and he has served on the editorial boards of Analytical Chemistry and Protein Science, among others.
Dr. Carr received his B.S. in 1976 from Union College and Ph.D. from MIT in 1980. After four years of postdoctoral training at Harvard Medical School and MIT, he joined SmithKline Pharmaceuticals (now GlaxoSmithKline), becoming director of Computational and Structural Sciences in 1997. Most recently he led protein science and proteomics groups at Millennium Pharmaceuticals in Cambridge, MA, prior to joining the Broad Institute in 2004.
Proteomic Technology Specialists
Steven Carr*, Leigh Anderson*, Amanda Paulovich*, Terry Pearson*, Christoph Borchers, Michael Gillette, Xiuwen Liu and Neal Gordon
Clinical Cancer Researchers
Peggy Porter, Constance Lehma, Julie Gralow
Bioinformaticians
Karl Clauser, ChenWei Lin
Biostatisticians
Steven Skates, David Ransohoff* and Pei Wang
Biologists
Bert Vogelstein, David Livingston, James Roberts, David Hockenberry, Tyler Jacks, Jonathan Pollack and Kornelia Polyak
* Denotes a member of the Internal Advisory Committee
Participating Institutions:
Broad Institute
Dana-Farber Cancer Research Institute
Fred Hutchinson Cancer Research Center
Epitomics, Inc.
Massachusetts General Hospital
Plasma Proteome Institute
University of Victoria
University of Victoria-Genome BC Proteomics Center
University of Washington
Epitome Biosystems
Other Essential Personnel:
Terri Addona, Susan Abbatiello, Eric Kuhn, Hasmik Keshishian, Michael Burgess, Diane Guay, JoAnn Lorenzo, Jeff Whitaker, Regine Schoenherr, Lei Zhao, Barbara Stein, Tanya Logvinenko, Nora Horick, Richard Morse, Timothy Nadler, James Graham, Christine Rauh-Adelmann, Angela Jackson, Matt Pope, Martin Soste, Derek Smith
About the Broad Institute:
The Broad Institute was founded in 2003 through the vision and generosity of Eli and Edythe Broad. The Broads recognized the inherent promise of genomics to change medicine and they saw the need to empower scientists to work together to realize that promise. Their founding gift was intended to provide the resources to build and demonstrate a new, collaborative model of science focused on transforming medicine. The Boston area’s pre-eminent biomedical research organizations – Harvard University and its affiliated hospitals, The Massachusetts Institute of Technology, and the Whitehead Institute for Biomedical Research – shared the Broads’ vision and joined together as the founding partners of the Broad Institute.
