Biological Interactions and Dynamics Publications

2009

Wu S, NM Lourette, N Tolic, R Zhao, R Robinson, AV Tolmachev, RD Smith, and L Pasa-Tolic. 2009. "An integrated top-down and bottom-up strategy for broadly characterizing protein isoforms and modifications." Journal of Proteome Research 8(3):1347-1357. Abstract We present an integrated top-down and bottom-up approach facilitated by concurrent liquid chromatography-mass spectrometry (LC-MS) analysis and fraction collection for comprehensive high-throughput intact protein profiling. The approach employs high resolution reversed phase (RP) LC separations coupled on-line with a 12T Fourier transform ion cyclotron resonance (FTICR) spectrometer to profile and tentatively identify modified proteins, using detected intact protein masses in conjunction with bare protein identifications from the bottom-up analysis of the same fraction. Selected identifications are incorporated into a target ion list for subsequent offline gas phase fragmentation that uses only an aliquot of the original fraction used for bottom-up analysis.
Lei C, D Hu, and EJ Ackerman. 2009. "Clay Nanoparticle-Supported Single-Molecule Fluorescence Spectroelectrochemistry." Nano Letters 9(2):655-658. Abstract We report single-molecule fluorescence spectroelectrochemistry on a clay-modified ITO electrode using cresyl violet as a redox fluorescent probe. Ensemble averaged experiments show that cresyl violet displays well-defined cyclic voltammograms when adsorbed on the clay-modified electrode. By probing the fluorescence intensity of a single cresyl violet molecule absorbed on clay surface, we can trace the redox reaction of individual molecules induced by the cyclic voltammetric potential scanning. Inhomogeneous interfacial electron transfer dynamics of the immobilized single cresyl violet molecules on the clay-modified surface were observed.
Tarasevich BJ, AS Lea, W Bernt, MH Engelhard, and WJ Shaw. 2009. "Adsorption of Amelogenin onto Self-Assembled and Fluoroapatite Surfaces." Journal of Physical Chemistry B 113(7):1833-1842. Abstract Abstract. The interactions of proteins at surfaces are of great importance to biomineralizaton processes and to the development and function of biomaterials. Amelogenin is a unique biomineralization protein because it self-assembles to form supramolecular structures called “nanospheres,” spherical aggregates of monomers that are 20-60 nm in diameter. Although the nanosphere quaternary structure has been observed in solution, the quaternary structure of amelogenin adsorbed onto surfaces is also of great interest because the surface structure is critical to its function. We report studies of the adsorption of the amelogenin onto self-assembled monolayers (SAMs) with COOH and CH3 end group functionality and single crystal fluoroapatite (FAP). Dynamic light scattering (DLS) experiments showed that the solutions contained nanospheres and aggregates of nanospheres. Protein adsorption onto the various substrates was evidenced by null ellipsometry, x-ray photoelectron spectroscopy (XPS), and external reflectance Fourier transform infrared spectroscopy (ERFTIR). Although only nanospheres were observed in solution, ellipsometry and atomic force microscopy (AFM) indicated that the protein adsorbates were much smaller structures than the original nanospheres, from monomers to small oligomers in size. Monomer adsorption was promoted onto the CH3 surfaces and small oligomer adsorption was promoted onto the COOH and FAP substrates. In some cases, remnants of the original nanospheres adsorbed as multilayers on top of the underlying subnanosphere layers. This work suggests that amelogenin can adsorb by the “shedding” or disassembling of substructures from the nanospheres onto substrates and indicates that amelogenin may have a range of possible quaternary structures depending on whether it is in solution or interacting with surfaces.
Ream TS, JR Haag, AT Wierzbicki, CD Nicora, AD Norbeck, JK Zhu, G Hagen, TJ Guilfoyle, L Pasa-Tolic, and CS Pikaard. 2009. "Subunit Compositions of the RNA-Silencing Enzymes Pol IV and Pol V Reveal Their Origins as Specialized Forms of RNA Polymerase II ." Molecular Cell 33(2):192-203. doi:doi:10.1016/j.molcel.2008.12.015 Abstract In addition to RNA polymerases I, II and III, which are multi-subunit RNA polymerases found in all eukaryotes, plants have catalytic subunits for two additional nuclear RNA polymerases, abbreviated as Pol IV and Pol V (formerly Pol IVa and Pol IVb, respectively). Pol IV and Pol V play non-redundant roles in siRNA-directed DNA methylation and gene silencing pathways.
Yang Z, ER Vorpagel, and J Laskin. 2009. "Influence of the Charge State on the Structures and Interactions of Vancomycin Antibiotics with Cell-Wall Analogue Peptides: Experimental and Theoretical Studies." Chemistry - a European Journal 15(9):2081-2090. doi:10.1002/chem.200802010 Abstract In this study we examined the effect of the charge state on the energetics and dynamics of dissociation of the non-covalent complex between the vancomycin and the cell wall peptide analogue Nα,Nε-diacetyl-L-Lys-D-Ala-D-Ala (V-Ac2KDADA). The binding energies between the vancomycin and the peptide were obtained from the RRKM modeling of the time- and energy resolved surface-induced dissociation (SID) experiments. Our results demonstrate that the stability of the complex toward fragmentation increases in the order: [V+Ac2KDADA+H]+2 < [V+Ac2KDADA+H]+ < [V+Ac2KDADA-H]-. Dissociation of the singly protonated and singly deprotonated complex is characterized by very large entropy effects indicating substantial increase in the conformational flexibility of the resulting products. The experimental threshold energies of 1.75 eV and 1.34 eV obtained for the [V+Ac2KDADA-H]- and [V+Ac2KDADA+H]+ , respectively, are in excellent agreement with the results of density functional theory (DFT) calculations. The increased stability of the deprotonated complex observed experimentally is attributed to the presence of three charged sites in the deprotonated complex as compared to only one charged site in the singly protonated complex. The low binding energy of 0.93 eV obtained for the doubly protonated complex suggests that this ion is destabilized by Coulomb repulsion between the singly protonated vancomycin and the singly protonated peptide comprising the complex.
Zhao R, SJ Ding, Y Shen, DG Camp, II, EA Livesay, HR Udseth, and RD Smith. 2009. "Automated metal-free multiple-column nanoLC for improved phosphopeptide analysis sensitivity and throughput." Journal of Chromatography B 877(8-9):663-670. Abstract We report on the development and characterization of an automated metal-free nanoscale multiple-capillary system for reversed-phase liquid chromatography-mass spectrometry analysis of phosphopeptides. The system incorporates a capillary column (50 um i.d. × 30 cm, packed with 5 um C18 particles) coupled on-line to a solid phase extraction column (150 um i.d. × 4 cm, packed with 5 um C18 particles). Electrospray ionization tips are constructed on the packed capillary column to couple the reversed-phase liquid chromatographic separation to a linear ion trap tandem mass spectrometer.
Boschek CB, DO Apiyo, TA Soares, HE Engelmann, N Pefaur, TP Straatsma, and CL Baird. 2009. "Engineering an ultra-stable affinity reagent based on Top7." Protein Engineering, Design & Selection 22(5):325-332. Abstract ABSTRACT Antibodies are widely used for diagnostic and therapeutic applications because of their sensitive and specific recognition of a wide range of targets; however, their application is limited by their structural complexity. More demanding applications require greater stability than can be achieved by immunoglobulin-based reagents. Highly-stable, proteinbased affinity reagents are being investigated for this role with the goal of identifying a suitable scaffold that can attain specificity and sensitivity similar to that of antibodies while performing under conditions where antibodies fail. We have engineered Top7 a highly stable, computationally-designed protein to specifically bind human CD4 by inserting a peptide sequence derived from a CD4-specific antibody. Molecular dynamics simulations were used to evaluate the structural effect of the peptide insertion at a specific site within Top7 and suggest that this Top7 variant retains conformational stability over 100 °C. This engineered protein specifically binds CD4 and, consistent with simulations, is extremely resistant to thermal and chemical denaturation retaining its secondary structure up to at least 95°C and requiring 6 M guanidine to completely unfold. This CD4-specific protein demonstrates the functionality of Top7 as a viable scaffold for use as a general affinity reagent which could serve as a robust and inexpensive alternative to antibodies.
Springer DL, JH Miller, SL Spinelli, L Pasa-Tolic, SO Purvine, DS Daly, RC Zangar, S Jin, N Blumberg, CW Francis, MB Taubman, AE Casey, SD Wittlin, and RP Phipps. 2009. "Platelet Proteome Changes Associated with Diabetes and During Platelet Storage for Transfusion." Journal of Proteome Research 8(5):2261-2272. Abstract Human platelets play a key role in homeostasis and thrombosis and have recently emerged as key regulators of inflammation. Platelets stored for transfusion produce pro-thrombotic and pro-inflammatory mediators implicated in adverse transfusion reactions. Correspondingly, these mediators are central players in pathological conditions including cardiovascular disease, the major cause of death in diabetics. In view of this, a mass spectrometry based proteomics study was performed on platelets collected from healthy and type-2 diabetics stored for transfusion. Strikingly, our innovative and sensitive proteomic approach identified 146 proteins that were either up- or down-regulated in type-2 diabetics relative to non-diabetic controls, 151 proteins whose abundances changed during a 5-day storage period and 22 proteins whose abundance changed after 5-days of storage were only observed in samples from diabetics. Notably our studies are the first to characterize the proteome of platelets from diabetics before and after storage for transfusion. These identified differences allow us to formulate new hypotheses and experimentation to improve clinical outcomes by targeting "high risk platelets" that render platelet transfusion less effective or even unsafe.
Umar AN, H Kang, AM Timmermans, MP Look, ME Meijer-van Gelder, MA den Bakker, N Jaitly, JW Martens, TM Luider, JA Foekens, and L Pasa-Tolic. 2009. "Identification of a putative protein profile associating with tamoxifen therapy resistance in breast cancer." Molecular & Cellular Proteomics. MCP 8(6):1278-1294. doi:10.1074/mcp.M800493-MCP200 Abstract Tamoxifen-resistance is a major cause of death in patients with recurrent breast cancer. Current clinical factors can correctly predict therapy response in only half of the treated patients. Identification of proteins that associate with tamoxifen-resistance is a first step towards better response prediction and tailored treatment of patients. In the present study we intended to identify putative protein biomarkers indicative of tamoxifen therapy-resistance in breast cancer, using nanoLC coupled with FTICR MS. Comparative proteome analysis was performed on ~5,500 pooled tumor cells (corresponding to ~550 ng protein lysate/analysis) obtained through laser capture microdissection (LCM) from two independently processed data sets (n=24 and n=27) containing both tamoxifen therapy-sensitive and therapy-resistant tumors. Peptides and proteins were identified by matching mass and elution time of newly acquired LC-MS features to information in previously generated accurate mass and time tag (AMT) reference databases.
Yang F, S Wu, DL Stenoien, R Zhao, ME Monroe, MA Gritsenko, SO Purvine, AD Polpitiya, N Tolic, Q Zhang, AD Norbeck, DJ Orton, RJ Moore, K Tang, GA Anderson, L Pasa-Tolic, DG Camp, II, and RD Smith. 2009. "Combined Pulsed-Q dissociation and electron transfer dissociation for identification and quantitation of iTRAQ–labeled phosphopeptides." Analytical Chemistry 81(10):4137-4143. doi:10.1021/ac802605m Abstract Multiplex isobaric tags for relative and absolute quantification (iTRAQ) enable high-throughput quantification of peptides via reporter ion signals in the low mass range of tandem mass spectra. A challenging but highly promising application is to analyze iTRAQ-labeled peptides using a sensitive linear ion trap mass spectrometer (LTQ-MS) and pulsed Q dissociation (PQD), a form of ion trap collision activated dissociation (CAD) designed to allow detection of low mass-to-charge fragment ions. Electron dissociation transfer (ETD), on the other hand, is complementary to PQD and is especially useful for sequencing peptides containing post-translational modifications (PTMs). Here, we developed an integrated workflow for robust and accurate quantitative identification of iTRAQ labeled phosphopeptides that integrates the PQD and ETD fragmentation methods together with PQD optimization, data management and bioinformatics tools. Analysis of the phosphoproteome of human fibroblast cells demonstrated that this hybrid mode is superior to either PQD or ETD alone for phosphopeptide identification and quantitation. The combined PQD/ETD approach can qualitatively identify additional phosphopeptides than ETD alone and PQD information can provide better quantitation of ETD identified iTRAQ-labeled phosphopeptides.

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