Conference Program - ABRF 2011 - Association of Biomolecular ...

Workshop SessionAbstractscase incorporation of the amino acid into protein must be its onlymetabolic fate within the muscle. The basic principle of measuringmuscle protein breakdown is to determine the rate at which intracellularamino acid tracer is diluted by the appearance of unlabeled amino acidthat is not coming from the plasma. There are different approachesto accomplishing this measurement, and the choice of the optimaldepends of the method used to measure protein synthesis. Ideally,methods to measure synthesis and breakdown are compatible (i.e., thesame units) to enable calculation of the balance between synthesis andbreakdown to determine if there is a net gain or loss in muscle protein.(W3-2) In Vivo Stable Isotope Labeling forQuantifying Amyloid-beta Kinetics in Alzheimer’sDisease: Is it All in our Head?K.E. YarasheskiBiomedical Mass Spectrometry Research Laboratory,Washington University School of Medicine, St. Louis, MO,United StatesMass spectrometry has revolutionized the manner in which we identify,characterize, and quantify proteins. In combination with in vivo stableisotope labeling strategies, mass spectrometry-based analyses canprovide valuable information about human amino acid and proteinkinetics, protein production (synthesis), and clearance (proteolysis)rates. Dysregulated or imbalanced protein synthesis and degradationrates is the basis for many clinical disorders. These protein kineticrates can be quantified in vivo and serve to identify potential targetsfor novel drug therapies. Our group uses an intravenous infusion of13C6-Leu, cerebral spinal fluid (CSF) sampling, affinity isolation ofrelevant proteins, and tandem mass spectrometry to quantify 13C6-Leu incorporation and removal rates from CSF amyloid-beta andapolipoproteins in Alzheimer’s disease patients. This approach hasidentified slower amyloid-beta clearance rates as a primary lesion thatmay explain an accumulation of amyloid plaques in Alzheimer’s disease.(W4) Insights for Expression of RecombinantProteins for Drug Target Validation(W4-2) Strategies for Optimized High-ThroughputCloning, Expression and Purification of RecombinantProteins in E. coliR. PageBrown University, Department of Molecular Biology, CellBiology and Biochemistry, Center for Genetics, Genomicsand Proteomics, Providence, RI, United StatesThe arrival of structural genomics, in addition to large-scale effortsinitiated in pharmaceutical companies, have resulted in the developmentof numerous new methods and strategies to minimize the time requiredto optimize the cloning, expression and purification of novel proteindrug targets. Here, I will present both best practices and unusual (‘lastditch’) methods that are used to successfully express and purify bothprokaryotic and eukaryotic proteins in E. coli. Recent developments inprotein expression, including co-expression with protein partners andbacterial chaperones will be presented. In addition, a comparison ofthe best solubility enhancing tags, and methods for subsequent tagremoval, will be presented. Finally, I will also describe two case studieswhich required highly tailored expression protocols for the productionof a bacterial toxin and eukaryotic phosphatase. In summary, the focusof this lecture will be to provide practical information that researchersactively involved in protein purification can readily implement intotheir own workflows. This work was supported in part by a medicalresearch grant from the American Cancer Society (RSG-08-067-01-LIB)and an NSF-CAREER award (MCB 0952550).(W5) Proteomics Tips and Tricks: FromDiscovery to Protein-Protein Interactions(W5-1) Proteomics Tips and Tricks: From Discoveryto Protein-Protein InteractionsM. CiliaUnited States Department of Agriculture/AgriculturalResearch Service, Robert W. Holley Center for Agricultureand Health, Cornell University, Ithaca, NY, United StatesProteins are the functional constituents of cells. The diverse physicalproperties of proteins enable them to be multifunctional and also posechallenges to analyzing their abundance, subcellular localization, andpotential interacting partners using proteomics. Some samples areespecially recalcitrant and pose additional challenges in proteomicanalysis due to the unanticipated modification of proteins. Thesechallenges extend from initial protein extraction to analysis usingmass spectrometry. Broadly, the focus of workshop will be to discussthe benefits and limitations of using proteomics to study proteinidentification, protein quantification, protein structure, and proteinproteininteractions. Examples will be drawn from different biologicaldisciplines, including cell biology, host-pathogen biology, anddevelopmental biology to demonstrate how scientists are solving theproblems posed by their discovery methods and biological systems.The workshop will include presentations from each panelist followedby an open discussion and question and answer session.(W5-2) Protein Interactions and Topologies in CellsJ. BruceUniversity of Washington, Department of Genome Sciences,Seattle, WA, United StatesLife on earth has evolved to utilize proteins as functional molecules dueto the wide diversity of structures and physical properties this class ofmolecules can exhibit. However, the divergent properties that criticallysupport life also pose fundamental challenges in all efforts to measurethe proteome. As a result, most proteomics experiments only samplea small subset of expressed molecules and typically appear biasedtowards cytoplasmic proteins. Furthermore, detection of proteinproteininteractions is even more challenging. For example, most largescalemethods such as the yeast two-hybrid, tandem affinity purification,and co-IP methods are predicated on the production/maintenanceof native protein structures and co-localization of native interactingpartners. These two requirements can result in failure to identify manybone fide interactions and in false discovery because many interactions46 • ABRF 2011 — Technologies to Enable Personalized Medicine