According to the National Cancer Institute, the greatest hope for the prevention and treatment of cancer exists in the understanding of molecular basis for disease initiation, progression and effective treatment based on the discovery of unique biomarkers. Cancer genomics provides much insight but it does not reveal the entire story.
“While very significant insights into biology are contained in gene sequencing and gene expression studies, research projects are more completely informed if they also have data on the ultimate products of gene expression – proteins and metabolites” said Arthur Moseley, director of the Proteomics Shared Resource.
The proteome refers to the complement of proteins, including their Post-Translational Modifications (PTMs), produced by an organism or a cellular system. This will vary with time and via circumstances that a cell or organism undergoes. Differential expression proteomics quantifies these changes in the proteome (both expression changes and changes in PTMs).
Surgical oncologist Rebekah “Becke” White, MD, utilizes the Proteomics Shared Resource. Her research focuses on gastrointestinal cancer.
“Aptamers are artificial oligonucleotides that bind to proteins with high affinity and specificity, analogous to antibodies,” White shared. “Aptamers are generated from large combinatorial libraries using iterative in vitro selection strategies against target proteins. We are using aptamers to probe complex mixtures of proteins, such as proteins secreted by pancreatic cancer cells, in order to identify novel biomarkers. Once we identify an aptamer with the desired properties, such as binding to the pancreatic cancer secretome, we need to identify its specific protein target.”
White believes the Proteomics Shared Resource is essential for the process.
“The protein of interest is typically isolated by a combination of biochemical separation of the secretome and affinity purification using the aptamer,” she said. “The Proteomics Shared Resource has helped us to troubleshoot the protein purification for subsequent mass spectrometric analysis and to interpret the resulting data. In our first pancreatic cancer selection, this analysis identified the aptamer target as Cyclophilin B, a protein that had not been evaluated as a secreted biomarker.”
The Proteomics Shared Resource provides qualitative/quantitative protein and metabolite analyses, using ultra-performance liquid chromatography and tandem mass spectrometry (UPLC/MS/MS) as its primary tool. Originally designed to provide all needed capabilities for mass spectrometry based proteomics for qualitative and quantitative analyses of simple or complex mixtures, including biomarker discovery and biomarker verification experiments, the resource’s services were expanded in 2013 to include qualitative and quantitative metabolite characterization.
For identification and biomarker discovery experiments (‘omic-scale qualitative and quantitative analyses), the laboratory is equipped with four high resolution accurate mass tandem mass spectrometers (MS/MS), each coupled to a dedicated UPLC or UPLC/UPLC system. Targeted quantitative experiments are performed on a UPLC/triple quadrupole tandem mass spectrometer using Multiple Reaction Monitoring. In addition, the laboratory has the tools needed for enrichments (chemical and/or antibody-based) of sub-proteomes based on Post-Translational Modifications, including phosphorylation, acetylation, ubiquitination, methylation, acylation, and S-nitrosylation. In 2014 the lab added a Hydrogen-Deuterium Exchange UPLC/MS/MS system for structural proteomics studies, complementing the traditional structural tools of X-Ray and NMR. Data processing is accomplished using two dedicated servers and a dedicated data storage system (two 36 TB data mirrors).
Established in 2007, the Duke Proteomic Shared Resource has performed more than 900 projects for more than 150 PIs. The lab includes Moseley, J. Will Thompson, PhD, associate director and assistant research professor; Laura Dubois, laboratory analyst II; Erik J. Soderblom, PhD, laboratory analyst II; Matthew Foster, PhD, assistant research professor; Meredith T. Mayer-Salman, research technician II; and Lisa St. John Williams, laboratory Analyst II. While the facility is available for use by the entire Duke research community, its CCSG support offers DCI members priority access.
“The Proteomics Resource makes expensive and sophisticated technologies accessible to all Duke Investigators in an efficient and cost-effective manner” Moseley said. “The experimental design of all experiments is created via collaborative discussions between shared resource senior staff and the investigators. This collaborative creation of the experimental design has proven to be essential, as it confirms that our staff have a clear understanding of the desired metrics of success for the biological/ clinical project. It also ensures that the biological/ clinical collaborators are fully informed as to the special requirements of the preparation of samples for UPLC/MS/MS analyses.”
Those utilizing the Proteomics Shared Resource depend on an expert team – knowledgeable and equally important, accessible.
“Art Moseley and his team have been extremely helpful,” said White. “Staff is accessible and always willing to answer questions. They respond quickly to emails, and we have met in person on several occasions to discuss more complex issues or ideas for new projects. Art and his team have helped us to put together methods for grants and manuscripts. We receive high-quality, prompt and reasonably priced services.”
The Proteomics Shared Resource, located in the Levine Science Research Center, is one of 13 shared resources offered by Duke to DCI researchers. Shared resources provide access to technologies, services and scientific consultation that enhance scientific interaction and productivity. Duke Cancer Institute members have priority access to the Proteomics Shared Resource. For more information, visit Proteomics or email Arthur Moseley.