PNNL-13501 - Pacific Northwest National Laboratory

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Figure 2. Hybridization of a cy3-labeled control RNA to a microarray with 1920 cDNA fragment to reveal immobilized polymerase chain reaction product analysis of peptide fragments that are representative of the complete protein sequence. To improve these methods, we developed a combination of protein digestion and gel extraction procedures that provide >95% coverage of a test protein. These procedures should be suitable for determining protein modifications with a high degree of confidence. Protein Arrays A major goal of this task is the development of protein arrays that will allow for the rapid identification and characterization of a large number of proteins involved in cell signaling pathways important in the genesis of ovarian cancer. In order to accomplish this, we are developing arrays of antibodies that will selectively bind cell-signaling proteins. Bound proteins will be evaluated using sophisticated mass spectrometric methods. This coupling of protein array technology with advanced mass spectrometric detection provides a high-throughput approach and a means to identify and characterize biologically important changes in the proteome (Figure 4). We have developed the initial methodologies necessary for the attachment of antibodies to a solid support that are compatible with mass spectrometry. Currently two different attachment chemistries have been employed. In both cases, a self-assembled monolayer is generated on gold, a surface that is preferred for MALDI-MS. The antibody is then covalently attached to the self-assembled monolayer. The first self-assembled monolayer we tried, dithiobis(succinimidyl propionate), is an amine-reactive, 50 FY 2000 <strong>Laboratory</strong> Directed Research and Development Annual Report homobifunctional cross-linker that covalently attaches the antibodies to the gold surface. We have also achieved good protein attachment using an alkanethiolated selfassembled monolayer. For this attachment strategy, a zero-length carbodiimide linker is employed to covalently link the terminal carboxylic acid group of the selfassembled monolayer with a primary amine from the protein. Additionally, we developed the necessary methods to analyze the different attachment chemistries both to monitor the attachment of the self-assembled monolayer and antibody and to address the functionality of the immobilized antibody. We are currently using these methods to optimize and extend our attachment strategies. Summary and Conclusions Technologies to characterize the relationship between signal transduction, environmental exposures, and human diseases are being implemented. These include • cDNA microarrays • antibodies to capture specific proteins • protein characterization by mass spectrometry • protein identification using bioinformatics programs. References Hobson et al. 2000. “Signal transduction mechanisms linking increased extracellular calcium to proliferation in ovarian surface epithelial cells.” Exp. Cell Res. 258:1-11. McNeil et al. 1998. “Functional calcium-sensing receptor expression in ovarian surface epithelial cells.” Amer. J. Obstet. Gynec. 178:305-313. Wong KK, RS Cheng, and SC Mok. “Identification of differentially over expressed genes in ovarian cancer cells by MICROMAX cDNA microarray.” BioTechniques (in press). Publications and Presentations Wong KK, RS Cheng, and SC Mok. “Identification of differentially over expressed genes in ovarian cancer cells by MICROMAX cDNA microarray.” BioTechniques (in press). Zangar RC, KL Wahl, CE Petersen, RD Rodland, DL Springer. 2000. “Protein primary structure determined by protease digestion and MALDI-MS peptide analysis.” To be presented at Hanford Symposium, Richland, Washington.
Immunoprecipitaton of tyrosinephosphorylated proteins Antibody A Immunoblot Anti-P-tyr Unk 1 2 3 B SDS-PAGE Stained gel Trypsin Digests MALDI-TOF Analysis 205 kDa 120 kDa 84 kDa 52 ProFound-SwissProt Analysis of Peptide Fragments Computer identification based on sizes of peptide fragments obtained from MALDI-MS Band Protein ID kDa Probability 1 IQ-GTPace activating protein 1 189 0.99 (mixed w/GAP-associated p190) 170 0.16 2 protein kinase C lambda 67 0.29 (mixture with EMH2, melanoma protein 60 0.29 3 blk [src kinase super-family] 57 >0.99 Figure 3. Identification of tyr-phosphorylated proteins using immunoprecipitation, gel separation, trypsin digestion and MALDI-MS Biosciences and Biotechnology 51
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Laboratory Directed Research and De
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Laboratory Directed Research and De
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Computational Science and Engineeri
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Policy and Management Sciences Asse
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Introduction Department of Energy O
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5. After reviews by the Technical a
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• The Technical Council peer revi
Page 15 and 16: methods applicable to combustion, s
Page 17 and 18: Summary Each national laboratory mu
Page 19 and 20: Study Control Number: PN0004/1411 A
Page 21 and 22: Table 1. Positive and negative ions
Page 23 and 24: m/z Arbitrary Units Figure 1. Mass
Page 25 and 26: introduce low-volatility compounds
Page 27 and 28: arrangement, but the charge is reta
Page 29 and 30: two liquid chromatographic gradient
Page 31 and 32: Integrated Microfabricated Devices
Page 33 and 34: Matrix Assisted Laser Desorption/Io
Page 35 and 36: Molecular Beam Synthesis and Charac
Page 37 and 38: temperature distribution of the des
Page 39 and 40: expected to result in significant a
Page 41 and 42: Study Control Number: PN99069/1397
Page 45 and 46: Seuss DT and KA Prather. 1999. “M
Page 47 and 48: Analysis of Receptor-Modulated Ion
Page 49 and 50: laboratory (Lu and Xie 1997; Lu et
Page 51 and 52: Automated DNA Fingerprinting Microa
Page 53 and 54: Comparison of 4 Xanthomonas Isolate
Page 55 and 56: Approach Hematopoietic (bone marrow
Page 59 and 60: Anti-Human lgG Human lgG Protein G
Page 61 and 62: Characterization of Global Gene Reg
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Page 65: identify novel proteins of importan
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Page 71 and 72: In the optical microscopy image, th
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Page 75 and 76: Figure 3. Hepa-1 cells undergoing a
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Page 91 and 92: Summary and Conclusions We demonstr
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Study Control Number: PN00021/1428
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Jones-Oliveira JB, JS Oliveira, HE
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• securely moves files to a targe
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Plenary invited lecture, “The Ele
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Figure 1. X3DGEN tetrahedral mesh o
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Figure 5a. OSO volume rendering of
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Development of Models of Cell-Signa
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SOS p 23 EGFR Professor Rodland at
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Oliveira JS, JB Jones-Oliveira, and
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Figure 2. Associating a dataset mar
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to 1) incorporate three-dimensional
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shown in Figure 1. Simulated result
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HostBuilder: A Tool for the Combina
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Invariant Discretization Methods fo
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Graphics, Inc., workstation. The co
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Mixed Hamiltonian Methods for Geoch
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guyanaite, and grimaldiite (see Fig
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in the Environmental Molecular Scie
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Simulation and Modeling of Electroc
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Summary and Conclusions We implemen
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Since the implementation of the gen
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asis of previous performance, perce
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Bioinformatics for High-Throughput
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Summary and Conclusions In previous
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User Cat - Supervisor (client) Anal
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The second goal was to research way
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Figure 1. A visualization technique
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and interactions. The flexibility o
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Figure 4. A filtered version of Fig
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Development of Modeling Tools for J
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Figure 2. Schematic of experimental
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Integrated Modeling and Simulation
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(a) (b) (c) (d) Figure 1. Results o
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Thurman DA. August 2000. Collaborat
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MARC Input initial m esh and result
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(Johnson 1999; Colligan 1999; Gould
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Modeling of High-Velocity Forming f
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Global divergence error 1 10 -14 0
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that users were unlikely to appreci
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Earth System Science
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the lateral extent of the plume so
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Biogeochemical Processes and Microb
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Analyses of populations of viable a
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The first task was to establish fiv
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purged for 2 minutes. The gas sampl
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developing code architecture to min
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Berkowitz, CM. June 2000. “Atmosp
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environmental monitoring, 2) portab
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corresponded to a current density o
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Enhancing Emissions Pre-Processing
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Environmental Fate and Effects of D
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nitrogen and unpredictable eutrophi
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Figure 1. Ordinary kriging of 2 m d
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precipitation of calcite occurred i
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Interrelationships Between Processe
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phenanthrene resistant fraction con
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injection of an immiscible gas phas
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still some key kinetic issues that
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Application of a Crop Model The res
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The Nature, Occurrence, and Origin
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Particle number concentration, 1/cm
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geometry optimized AlOOH and FeOOH
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allowable parameters using thermody
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TCE + 3H + + 3Fe 2+ Fe)=> acetylene
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Use of Shear-Thinning Fluids for In
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concentration of 0.5% AMX was the m
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Roberts AL, LA Totten, WA Arnold, D
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tetra-scale computing, envisioned a
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Energy Technology and Management
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Figure 2. Setup for the second expe
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phase. The algorithms will be teste
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[Pb-212]/[Pb-212] final 100 10 1 0.
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Plasma Particle Dielectric Fiber El
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(species, sub-species, and sex), sp
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Figure 3. Relative risk of bone and
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Development of a Preliminary Physio
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Development of a Virtual Respirator
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Figure 3. Use of the chest cavity a
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Examination of the Use of Diazolumi
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Indoor Air Pathways to Nuclear, Che
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Source Building Release Observed re
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To illustrate the potential impact
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Non-Invasive Biological Monitoring
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Materials Science and Technology
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Figure 1. (a) - (c) Successive magn
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Component Fabrication and Assembly
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Development of a Low-Cost Photoelec
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elaxations, indicating the adequacy
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Promising chemical durability, as m
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Study Control Number : PN98028/1274
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High Power Density Solid Oxide Fuel
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Hybrid Plasma Process for Vacuum De
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Matson DW, PM Martin, WD Bennett, J
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We showed the proof-of-principle ap
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Bandgap Energy (eV) 2.9 2.85 2.8 2.
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Ultrathin Electrolyte Test Results
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Detailed Investigations on Hydrogen
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identified low energy step structur
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Development of Novel Photo-Catalyst
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-2 -1 Energy (eV) 0 1 2 Cu2O SrTiO3
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Here, we perform adsorption and des
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exceed those in the all-metal devic
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Intensity (a.u.) 0 100 200 300 400
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integrated voltage (V) 0.10 0.08 0.
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The first step was to implement thi
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Spontaneous Formation of Cu2O Nano-
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Actinide Chemistry at the Aqueous-M
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Figure 5. Close-up atomic force mic
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to 300°C and 400°C. Unfortunately
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Summary and Conclusions Transmutati
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Advanced Calibration/Registration T
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Figure 1a. Violet filter Figure 1b.
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Autonomous Ultrasonic Probe for Pro
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containing nitrous oxide, an optica
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Chemical Detection Using Cavity Enh
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Figure 3 is a color rendered simula
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appropriate study has been performe
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Exploitation of Conventional Chemic
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Figures 3 and 4 illustrate the impa
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enefits of using a modified spread-
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Single Channel Signal 0.0025 0.0020
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Therefore, in order to extract the
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A next-generation technology is nee
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Wind Dir. Figure 2. Negative ion co
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Volumetric Imaging of Materials by
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amplitude and peak frequency change
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Figure 3. 13 C NMR spectrum of corn
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Approach Two flow loops, one that o
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Modification of Ion Exchange Resins
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Permanganate Treatment for the Deco
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minimized by medium exchange. After
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Figure 1. Thermogravimetric analysi
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Validation and Scale-Up of Monosacc
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Concentration (g/100g solution) Con
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Analysis of High-Volume, Hyper-Dime
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ecent figure of merit values. Shoul
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Probabilistic Methods Development f
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a) Traditional PCA-based process co
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Acronyms and Abbreviations 2-D two-
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PNNL-13501 - Pacific Northwest National Laboratory

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