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

Laboratory is located next door and works closely with the ProteomicsCenter. Their mass spectrometers includes Applied Biosystems-MDSSciex 4000 Q Trap with Autosampler; Applied Biosystems Q-Star EliteQuad-TOF Hybrid LC/MS/MS system; Applied Biosystems Voyager-DE STR MALDI-TOF; Thermo Finnigan LCQ Deca Ion Trap MS withAutosampler and PDA; Agilent 6890-5975 GC-MS with Autosamplerand Waters LCT Premier with ACQUITY UPLC and Autosampler.184 New MAbPac Phases for MonoclonalAntibody (MAb) Variant AnalysisG. Gendeh, S. Rao, Y.X. Hou, X. Liu, Y. Agroskin,C. PohlDionex Corporation, Sunnyvale, CA, United States182 Microscale Thermophoresis:Interactions of Proteins, SmallMolecules, Nucleic Acids, and VesiclesS. Duhr, P. BaaskeNanoTemper Technologies GmbH, Munich,GermanyThis work gives an overview on a new Technology for the measurementof biomolecule interaction that is termed Microscale Thermophoresis(MST). The term Microscale Thermophoresis refers to the directedmovement of molecules in optically generated microscopic temperaturegradients. This thermophoretic movement is determined by the entropyof the hydration shell around the molecules. Almost all interactionsbetween molecules and virtually any biochemical process related toa change in size, stability and conformation of molecules alters thishydration shell and can be quantified. Such changes allow quantificationof binding affinities of proteins, nucleic acids and small molecules aswell as measurement of enzymatic activities with MST. In additionalso functional studies of small molecule inhibitors are possible. Themicroscopic temperature gradient is generated by an IR-Laser, whichis strongly absorbed by water. The readout method of the interactionanalysis is based on fluorescence: intrinsic fluorescence of proteins canbe used as well as proteins expressed with GFP/YFP/RFP and also dyelabeled biomolecules. In this presentation we will describe the technicaldetails and the benefits of the Microscale Thermophoresis technologyplatform. We will show examples for interaction measurements rangingfrom protein -ribosome, protein -protein, small molecule -receptorbinding to studies where the interactions between receptor containingvesicles and proteins are analyzed.183 Reduction of Sample Carryover inProteomics LC-MS ExperimentsG. Gendeh, M. Karsten, E.J. Sneekes, R. SwartDionex Corporation, Sunnyvale, CA, United StatesRelevant biomarkers are often present at concentrations near or belowthe detection limit of current analytical methods. Despite this challenge,several biomarker candidates have been identified and moved into thevalidation phase. The increase in sensitivity of analytical methods andmass spectrometry, in particular, over the past years is the reason forthis accomplishment. However, a sensitivity increase alone is insufficientto accurately identify potential biomarkers; carryover reduction is alsoimportant to ensure a marker is actually present in the sample beinganalyzed. Therefore, reduction of carryover has received increasedattention from the proteomics community.MAbs generally exhibit complex heterogeneity including glycosylation,oxidation, phosphorylation, amino-terminal modifications, incompleteprocessing of the C-terminus, and asparagine deamidation. Thesevariations in composition could impact their efficacy, stability, and safety.Monitoring and reporting such variations in therapeutic proteins isrequired by the FDA and other regulatory agencies. Two new MAbPac TMphases were developed to meet these needs. The MAbPac SCX-10 is anewly designed strong cation-exchange column for the characterizationof heterogeneity of MAbs. This is a complementary addition to theexisting ProPac® WCX-10 column that provides high resolution andorthogonal selectivity for MAb charge variant analysis. The MAbPacSCX stationary phase is based on nonporous, highly cross-linked styrenictype polymeric media with a proprietary hydrophilic coating. Sulfonicacid functionality is added through controlled radical polymerizationgrafting. These particles exhibit a wide range of pH stability with highselectivity and minimal band spreading. The MAbPac SEC-1 is a newsize-exclusion chromatography (SEC) column specifically developed forcharacterization of monoclonal antibody (MAb) aggregates, enzymedigested fragments, and other size-based separation applications. TheMAbPac SEC column is based on high-purity, spherical, porous (300Å), 5 μm silica covalently modified with a proprietary diol hydrophiliclayer. This stationary phase can handle both high- and low- salt eluentsas well as mass spectrometry compatible eluents. The MAbPac columnis packed into a nonmetallic, biocompatible PEEK TM column housingto eliminate metal contamination from the column hardware that cancompromise MAb separations. The stationary phase is designed tominimize undesired nonspecific interactions between proteins and thestationary phase. Various applications with relevant comparisons alongwith a demonstration of the ruggedness of these new phases are shownin this poster.185 The VGN Proteomics Module: ATransferable Laboratory Module forUndergraduatesJ. MurrayVermont Genetics Network, University of Vermont,Burlington VT, United StatesThe Vermont Genetics Network (VGN) Outreach Core’s mission isto bring cutting-edge technology and knowledge to undergraduatesat colleges throughout the state of Vermont. The VGN ProteomicsOutreach project initiated in the fall of 2009 exposes undergraduatesin the state of Vermont to proteomics technology using handsonlaboratory experiences. We provide all teaching materials,laboratory materials and if necessary equipment for colleges withinthe state to run the module. All materials become the property of therecipient institution upon completion of the laboratory module. Theundergraduate students learn about this cutting edge technology andgain new skills that we believe will help them with their future scientificcareers. In this transferable module, students learn how proteinexpression in yeast is changed after exposure to oxidative stress oran environmental toxin. Total protein is then harvested and preparedfor 2D gel analysis. Proteins with differential expression are isolatedfrom the 2D gel and prepared for Mass Spectrometry at the UVMPoster AbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 83