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

are the best methods for optimal recovery and proteome coverage.A novel approach for fractionating samples at the level of proteincomplexes will also be discussed.(S6-2) Improving the Comprehensiveness of Large-Scale Proteomics Experiments Using AdvancedComputational Tools and Accurate MultipleHypothesis Testing StatisticsM.J. MacCoss, J. Egertson, B. Frewen, L. Käll, W. NobleDepartment of Genome Sciences, University of Washington,Seattle, WA, United StatesMass spectrometry based technology for the analysis of complexprotein mixtures has improved at an amazing rate. With each newinstrument release, mass spectrometers have become more sensitiveand have faster MS/MS data acquisition speeds. Furthermore,instruments are continuously improving the dynamic range, massaccuracy, and resolution of the resulting mass spectrometry data. Allof these developments have increased the number of peptides thatcan be identified and quantified without extending the overall analysistime. While the technological hardware advances that are required toincrease the number of peptide identifications by 50% with a constantanalysis time is monumental, we have been able to demonstrate thatincrease in performance without increasing the analysis time at all. Toaccomplish this, we have made use of improved database searchingalgorithms, spectrum library searching, use of chromatographicretention time, powerful machine learning tools, accurate multiplehypothesis testing statistics, and many more. Strategies will bediscussed on how to increase the comprehensiveness of any datasetusing improved data analysis strategies.(S6-3) In-Depth Analysis of Human and MousePlasma Using 3-D And 4-D Fractionation StrategiesD.W. SpeicherThe Wistar Institute, Philadelphia, PA, United StatesIn-depth profiling of plasma proteomes can potentially identify noveldisease biomarkers. But few biomarkers identified by proteomicapproaches have advanced to early-stage clinical testing because theyoften are not sufficiently disease specific. Major challenges in plasmaproteome analysis include the very wide dynamic range of proteinconcentrations, the high protein complexity, and the substantialheterogeneity of most protein concentrations in the normal humanpopulation. Because most disease-specific biomarkers are present inblood at very low concentrations, extensive fractionation is requiredprior to LC-MS/MS analysis. In general, more fractionation will result ingreater depth of analysis, but there is a point of diminishing return foreach fractionation method and throughput decreases as the numberof LC-MS/MS runs per proteome increases. A common feature of mostcurrent plasma profiling methods is to first immunodeplete as manyhigh abundance plasma proteins as possible, followed by extensiveprotein fractionation of the depleted plasma prior to trypsin digestionand LC-MS/MS. In addition, reliable quantitative comparisons areneeded for most types of studies. While all quantitative methods havestrengths and weaknesses, label-free quantitative comparison of LC-MSsignals is increasing in popularity and seems adequately reproduciblefor most studies. Our laboratory commonly uses two alternative plasmaproteome analysis strategies. One powerful approach utilizes a 3-Dprotein/peptide profiling method consisting of depleting 20 abundantproteins followed by 1-D SDS PAGE, fractionation of the gel lane into20 to 60 fractions and LC-MS/MS analysis. Proteins can be quantitativelycompared using label-free analysis of ion current patterns from the MSfull scans. An even greater depth of analysis can be achieved using a4-D protein/peptide profiling strategy utilizing microscale solutionisoelectrofocusing of proteins prior the SDS gel in the 3-D scheme,although throughput is substantially reduced.(S7) Next Generation Sequencing Technologieson the Horizon(S7-1) Towards Optical DNA Sequencing UsingNanopore ArraysA. MellerDepartment of Biomedical Engineering, Boston University,Boston, MA, United StatesNext generation DNA sequencing methods that utilize nanometersizepores have been subject of numerous studies in past years. Oneof the compelling features of the nanopore technique lies in its abilityto electrophoretically focus long DNA strands towards the pore areaand thread the molecules inside the pore in a highly efficient manner.Thus extremely small copy numbers of the target DNA are requiredfor analyses†, circumventing the need for costly and time-consumingtarget amplification. One of the major bottlenecks for the realizationof a viable nanopore-based DNA sequencing has been the ability tosimultaneously read the electrical signals from hundreds to thousandsof nanopores densely fabricated on sub-millimeter size silicon chip. Toaddress this issue we develop an extremely high throughput singlemoleculeDNA sequencing technique, which employs optical, widefieldreadout from DNA molecules electrically mobilized throughthe nanopores. Our method consists of two steps: First, target DNAmolecules are converted according to pre-determined code, which isrecognized by molecular beacons with four types of fluorophores (eachuniquely corresponding to one of the four DNA bases). Solid-statenanopores are then used to sequentially strip off the beacons, leadingto a series of photon bursts that can be detected with a custom mademicroscope. Notably the method circumvents the use of enzymes in thereadout stage, and is thus not affected by their limited processivity andlifetime. Here we demonstrate the feasibility of this method using a twocolor model system, and show for the first time, individual nucleotiderecognition from multiple nanopores simultaneously‡, allowingstraightforward parallelization of our system to nanopore arrays. †.Wanunu, M., W. Morrison, Y. Rabin, A. Y. Grosberg, and A. Meller. 2010.Electrostatic Focusing of Unlabeled DNA into Nanoscale Pores using aSalt Gradient. Nature Nanotechnology 5:160-165. ‡. McNally, B., A.Singer, Z. Yu, Y. Sun, Z. Weng, and A. Meller. 2010. Optical Recognitionof Converted DNA Nucleotides for Single-Molecule DNA SequencingUsing Nanopore Arrays. Nano Letters 10:2237-2244.Scientific SessionAbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 41