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

Scientific SessionAbstracts(S7-2) Single Molecule Real-Time DNA Sequencingon the Surface of a Quantum-Dot NanocrystalP.B. Vander HornGenetic Systems, Life Technologies, Carlsbad, CA, UnitedStatesA single molecule, long read-length, real-time sequencingby-synthesistechnology has been developed by buildinga sequencer directly on the surface of a ~ 10 nm quantumdotnanocrystal. Fluorescence resonance energy-transfertechnology (FRET) is utilized for DNA sequence detection, inwhich signals from the quantum-dot labeled DNA polymeraseplus 4 DNA-base-specific acceptor dyes are simultaneouslydetected. Precisely engineered sequencing-grade Qdot TMnanocrystals are smaller than current commercially availablematerials (to increase FRET signals), and have an exctinctioncoefficient ~100X greater than organic-dyes, allowing for verylow levels of excitation power to be used while sequencing,Acting as the FRET donor, the Qdot TM -polymerase generates acorrelated “photon-dip” for every inserted based (termed the“quantum-correlation-signal”), allowing for more accurate basecalling.Because the sequencer is not physically bound to anysolid substrate, it can be exchanged (like a reagent) during midsequenceruns, effectively replacing damaged non-functioningpolymerases mid-reaction. Each exchange cycle lengthens theeffective read-length of the sequencer. In this manner, theread-length can be continuously extended without “gaps”.Expanding upon this flexibility, after sequencing a particularlength of DNA, the newly synthesized strand can be selectivelyremoved. The original genomic DNA strand is then re-primed,Qdot TM -polymerase sequencers are rebound, and the identicalgenomic DNA strand can be sequenced again, greatly increasingthe net accuracy and not requiring circularization of genomictemplates. In combining these features, the desired accuracyand read-length can be “tuned” by adjusting the number ofreagent exchange cycles. Because each sequencing reaction canbe completed in minutes, multiple exchange experiments canbe performed per sequencing hour. These Qdot TM -polymerasesequencers can also bind to ultra-long DNA segments (>10kb)at multiple positions along the length of the DNA andsequence while moving “horizontally” (parallel to TIRF field),enabling the possibility of “ordered-reads” for long-phasedhaplotype sequencing. Examples of real-time sequencing ofhomopolymeric, patterned, and complex templates will beshown.(S9) Proteomics StandardizationD.B. FriedmanProteomics Laboratory Mass Spectrometry Research Center,Vanderbilt University, Nashville, TN, United StatesThe needs and issues related to establishing inter-laboratorystandardization in quantitative proteomics will be highlighted bypresentations from three international initiatives. By establishing theneed for standardization, each group will also highlight the tremendousamount of instrument and experimental variation that is also measuredwhen trying to determine real biological changes. The Spanish-basedProteoRed consortium addresses multiple proteomics platforms,ranging from MS-based to gel-based. The NIH CPTAC network isfocused on MS-based studies, and the UK-based FixingProteomicsinitiative covers mostly gel-based methods. Results from thesestudies will demonstrate complement and overlap in technology andapproaches used between these groups, all motivated by the universalgoal of standardizing these complex technologies across laboratories.(S9-1) A Multi-Laboratory Study AssessingRobustness and Reproducibility of PlasmaReference Sample for Benchmarking LC-MS PlatformPerformanceJ.P. Albar 5 , A. Campos 1,4 , E. Oliveira 1,4 ,S. Martínez-Bartolomé 3,4 , F. Canals 2,41Barcelona Science Park, Barcelona, Spain; 2 Vall d’HebronUniversity Hospital Research Institute, Barcelona, Spain;3Centro Nacional de Biotecnologia-CSIC, Madrid, Spain;4ProteoRed Consortium, Spanish National Institute ofProteomics, Madrid, Spain; 5 ProteoRed, National Center forBiotechnology-CSIC, Madrid, SpainAn increasingly common request for proteomics core facilities isdetermining qualitative and quantitative differences among clinicalsamples such as plasma, CSF, or urine. One of the missions of theSpanish Network of Proteomics Facilities (ProteoRed-ISCIII) is to assistits proteomics core facilities in evaluating their capabilities to performqualitative and quantitative proteomics analysis. This year, in an attemptto represent a realistic experiment scenario that might be requested toa proteomics core facility, we provided a moderately complex plasmastandard reference sample to be used for routine QC monitoring oflaboratory instrumentation. The ProteoRed Plasma Reference (PPR)sample is a subset of highly abundant well-characterized human plasmaproteins with a number of isoforms, in addition to 4 spiked-in proteins,altogether distributed over 5 orders of magnitude in concentration.The PPR sample was recently stress tested in the latest ProteoRedMulticenter Experiment (PME6) that counted with the participationof 17 proteomics facilities using a wide range of LC-MS platforms. Werequested the sample be analyzed in a single LC-MS run in experimentaltriplicate (3 different digestions). Evaluation of the results submitted bythe study participants revealed moderate discrepancies at the peptideidentification level, and poor overlap at the protein identificationlevel. In an attempt to identify the source of such irreproducibility,raw data of 8 laboratories (24 LC-MS runs) were reanalyzed centrallyusing a standardized data analysis pipeline, which included proteininference using ProteinProphet software. We found that the majorityof protein identification discrepancies across submitted reports ofthese 8 laboratories were due to inconsistencies on how data analysisand computational tools group and/or infer proteins. Immunoglobulin42 • ABRF 2011 — Technologies to Enable Personalized Medicine