17 results on '"Clarke, David J."'
Search Results
2. Interlaboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry.
- Author
-
Srzentić K, Fornelli L, Tsybin YO, Loo JA, Seckler H, Agar JN, Anderson LC, Bai DL, Beck A, Brodbelt JS, van der Burgt YEM, Chamot-Rooke J, Chatterjee S, Chen Y, Clarke DJ, Danis PO, Diedrich JK, D'Ippolito RA, Dupré M, Gasilova N, Ge Y, Goo YA, Goodlett DR, Greer S, Haselmann KF, He L, Hendrickson CL, Hinkle JD, Holt MV, Hughes S, Hunt DF, Kelleher NL, Kozhinov AN, Lin Z, Malosse C, Marshall AG, Menin L, Millikin RJ, Nagornov KO, Nicolardi S, Paša-Tolić L, Pengelley S, Quebbemann NR, Resemann A, Sandoval W, Sarin R, Schmitt ND, Shabanowitz J, Shaw JB, Shortreed MR, Smith LM, Sobott F, Suckau D, Toby T, Weisbrod CR, Wildburger NC, Yates JR 3rd, Yoon SH, Young NL, and Zhou M
- Subjects
- Animals, Complementarity Determining Regions analysis, Complementarity Determining Regions chemistry, Complementarity Determining Regions genetics, Humans, Mice, Antibodies, Monoclonal analysis, Antibodies, Monoclonal chemistry, Antibodies, Monoclonal genetics, Mass Spectrometry methods, Proteomics methods
- Abstract
The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product's primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information on the common TD/MD MS methods and practices in the field. A panel of three mAbs was selected and centrally provided to 20 laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). Various MS instrument platforms and ion dissociation techniques were employed. The present study confirms that TD/MD MS tools are available in laboratories worldwide and provide complementary information to the BU approach that can be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. A primary limitation revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS.
- Published
- 2020
- Full Text
- View/download PDF
3. Isotope Depletion Mass Spectrometry (ID-MS) for Accurate Mass Determination and Improved Top-Down Sequence Coverage of Intact Proteins.
- Author
-
Gallagher KJ, Palasser M, Hughes S, Mackay CL, Kilgour DPA, and Clarke DJ
- Subjects
- Amino Acid Sequence, Animals, Bacterial Proteins chemistry, Carbonic Anhydrases chemistry, Cattle, Ferritins chemistry, Fourier Analysis, Models, Molecular, Proteomics, Rhodospirillum rubrum chemistry, Sphingomonas chemistry, Mass Spectrometry methods, Proteins chemistry, Sequence Analysis, Protein methods
- Abstract
Top-down mass spectrometry (MS) is an increasingly important technique for protein characterization. However, in many biological MS experiments, the practicality of applying top-down methodologies is still limited at higher molecular mass. In large part, this is due to the detrimental effect resulting from the partitioning of the mass spectral signal into an increasing number of isotopic peaks as molecular mass increases. Reducing the isotopologue distribution of proteins via depletion of heavy stable isotopes was first reported over 20 years ago (Marshall, A. G.; Senko, M. W.; Li, W.; Li, M.; Dillon, S., Guan, S.; Logan, T. M.. Protein Molecular Mass to 1 Da by
13 C,15 N Double-Depletion and FT-ICR Mass Spectrometry. J. Am. Chem. Soc. 1997, 119, 433-434.) and has been demonstrated for several small proteins. Here we extend this approach, introducing a new highly efficient method for the production of recombinant proteins depleted in13 C and15 N and demonstrating its advantages for top-down analysis of larger proteins (up to ∼50 kDa). FT-ICR MS of isotopically depleted proteins reveals dramatically reduced isotope distributions with monoisotopic signal observed up to 50 kDa. In top-down fragmentation experiments, the reduced spectral complexity alleviates fragment-ion signal overlap, the presence of monoisotopic signals allows assignment with higher mass accuracy, and the dramatic increase in signal-to-noise ratio (up to 7-fold) permits vastly reduced acquisition times. These compounding benefits allow the assignment of ∼3-fold more fragment ions than comparable analyses of proteins with natural isotopic abundances. Finally, we demonstrate greatly increased sequence coverage in time-limited top-down experiments-highlighting advantages for top-down LC-MS/MS workflows and top-down proteomics.- Published
- 2020
- Full Text
- View/download PDF
4. Insights into the conformations of three structurally diverse proteins: cytochrome c, p53, and MDM2, provided by variable-temperature ion mobility mass spectrometry.
- Author
-
Dickinson ER, Jurneczko E, Pacholarz KJ, Clarke DJ, Reeves M, Ball KL, Hupp T, Campopiano D, Nikolova PV, and Barran PE
- Subjects
- Animals, DNA metabolism, Models, Molecular, Protein Structure, Tertiary, Tumor Suppressor Protein p53 metabolism, Cytochromes c chemistry, Mass Spectrometry methods, Proto-Oncogene Proteins c-mdm2 chemistry, Temperature, Tumor Suppressor Protein p53 chemistry
- Abstract
Thermally induced conformational transitions of three proteins of increasing intrinsic disorder-cytochrome c, the tumor suppressor protein p53 DNA binding domain (p53 DBD), and the N-terminus of the oncoprotein murine double minute 2 (NT-MDM2)-have been studied by native mass spectrometry and variable-temperature drift time ion mobility mass spectrometry (VT-DT-IM-MS). Ion mobility measurements were carried out at temperatures ranging from 200 to 571 K. Multiple conformations are observable over several charge states for all three monomeric proteins, and for cytochrome c, dimers of significant intensity are also observed. Cytochrome c [M + 5H](5+) ions present in one conformer of CCS ∼1200 Å(2), undergoing compaction in line with the reported Tmelt = 360.15 K before slight unfolding at 571 K. The more extended [M + 7H](7+) cytochrome c monomer presents as two conformers undergoing similar compaction and structural rearrangements, prior to thermally induced unfolding. The [D + 11H](11+) dimer presents as two conformers, which undergo slight structural compaction or annealing before dissociation. p53 DBD follows a trend of structural collapse before an increase in the observed collision cross section (CCS), akin to that observed for cytochrome c but proceeding more smoothly. At 300 K, the monomeric charge states present in two conformational families, which compact to one conformer of CCS ∼1750 Å(2) at 365 K, in line with the low solution Tmelt = 315-317 K. The protein then extends to produce either a broad unresolved CCS distribution or, for z > 9, two conformers. NT-MDM2 exhibits a greater number of structural rearrangements, displaying charge-state-dependent unfolding pathways. DT-IM-MS experiments at 200 K resolve multiple conformers. Low charge state species of NT-MDM2 present as a single compact conformational family centered on CCS ∼1250 Å(2) at 300 K. This undergoes conformational tightening in line with the solution Tmelt = 348 K before unfolding at the highest temperatures. The more extended charge states present in two or more conformers at room temperature, undergoing thermally induced unfolding before significant structural collapse or annealing at high temperatures. Variable-temperature IM-MS is here shown to be an exciting approach to discern protein unfolding pathways for conformationally diverse proteins.
- Published
- 2015
- Full Text
- View/download PDF
5. Mapping a noncovalent protein-peptide interface by top-down FTICR mass spectrometry using electron capture dissociation.
- Author
-
Clarke DJ, Murray E, Hupp T, Mackay CL, and Langridge-Smith PR
- Subjects
- Amino Acid Sequence, Binding Sites, Electrons, Humans, Molecular Sequence Data, Mucoproteins, Oncogene Proteins, Peptide Fragments metabolism, Protein Binding, Proteins metabolism, Fourier Analysis, Mass Spectrometry methods, Peptide Fragments chemistry, Protein Interaction Mapping methods, Proteins chemistry
- Abstract
Noncovalent protein-ligand and protein-protein complexes are readily detected using electrospray ionization mass spectrometry (ESI MS). Furthermore, recent reports have demonstrated that careful use of electron capture dissociation (ECD) fragmentation allows covalent backbone bonds of protein complexes to be dissociated without disruption of noncovalent protein-ligand interactions. In this way the site of protein-ligand interfaces can be identified. To date, protein-ligand complexes, which have proven tractable to this technique, have been mediated by ionic electrostatic interactions, i.e., ion pair interactions or salt bridging. Here we extend this methodology by applying ECD to study a protein-peptide complex that contains no electrostatics interactions. We analyzed the complex between the 21 kDa p53-inhibitor protein anterior gradient-2 and its hexapeptide binding ligand (PTTIYY). ECD fragmentation of the 1:1 complex occurs with retention of protein-peptide binding and analysis of the resulting fragments allows the binding interface to be localized to a C-terminal region between residues 109 and 175. These finding are supported by a solution-phase competition assay, which implicates the region between residues 108 and 122 within AGR2 as the PTTIYY binding interface. Our study expands previous findings by demonstrating that top-down ECD mass spectrometry can be used to determine directly the sites of peptide-protein interfaces. This highlights the growing potential of using ECD and related top-down fragmentation techniques for interrogation of protein-protein interfaces.
- Published
- 2011
- Full Text
- View/download PDF
6. Identification of two reactive cysteine residues in the tumor suppressor protein p53 using top-down FTICR mass spectrometry.
- Author
-
Scotcher J, Clarke DJ, Weidt SK, Mackay CL, Hupp TR, Sadler PJ, and Langridge-Smith PR
- Subjects
- Alkylating Agents, Amino Acid Sequence, Crystallography, X-Ray, Cysteine metabolism, Ethylmaleimide, Fourier Analysis, Humans, Models, Molecular, Molecular Sequence Data, Oxidation-Reduction, Peptide Fragments chemistry, Peptide Fragments metabolism, Protein Unfolding, Temperature, Tumor Suppressor Protein p53 metabolism, Cysteine chemistry, Mass Spectrometry methods, Tumor Suppressor Protein p53 chemistry
- Abstract
The tumor suppressor p53 is a redox-regulated transcription factor involved in cell cycle arrest, apoptosis and senescence in response to multiple forms of stress, as well as many other cellular processes such as DNA repair, glycolysis, autophagy, oxidative stress and differentiation. The discovery of cysteine-targeting compounds that cause re-activation of mutant p53 and the death of tumor cells in vivo has emphasized the functional importance of p53 thiols. Using a combination of top-down and middle-down FTICR mass spectrometry, we show that of the 10 Cys residues in the core domain of wild-type p53, Cys182 and Cys277 exhibit a remarkable preference for modification by the alkylating reagent N-ethylmaleimide. The assignment of Cys182 and Cys277 as the two reactive Cys residues was confirmed by site-directed mutagenesis. Further alkylation of p53 beyond Cys182 and Cys277 was found to trigger co-operative modification of the remaining seven Cys residues and protein unfolding. This study highlights the power of top-down FTICR mass spectrometry for analysis of the cysteine reactivity and redox chemistry in multiple cysteine-containing proteins., (© American Society for Mass Spectrometry, 2011)
- Published
- 2011
- Full Text
- View/download PDF
7. Autopiquer - a Robust and Reliable Peak Detection Algorithm for Mass Spectrometry
- Author
-
Kilgour, David P. A., Hughes, Sam, Kilgour, Samantha L., Mackay, C. Logan, Palmblad, Magnus, Tran, Bao Quoc, Goo, Young Ah, Ernst, Robert K., Clarke, David J., and Goodlett, David R.
- Published
- 2017
- Full Text
- View/download PDF
8. A native mass spectrometry platform identifies HOP inhibitors that modulate the HSP90–HOP protein–protein interaction.
- Author
-
Veale, Clinton G. L., Mateos-Jiménez, Maria, Vaaltyn, Michaelone C., Müller, Ronel, Makhubu, Matodzi P., Alhassan, Mahama, de la Torre, Beatriz G., Albericio, Fernando, Mackay, C. Logan, Edkins, Adrienne L., and Clarke, David J.
- Subjects
PROTEIN-protein interactions ,MASS spectrometry ,PROOF of concept ,PEPTIDES - Abstract
Herein we describe a native mass spectromery protein–peptide model as a competent surrogate for the HOP–HSP90 protein–protein interaction (PPI), application of which led to the qualititive identification of two new peptides capable of in vitro PPI disruption. This proof of concept study offers a viable alternative for PPI inhibitor screening. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
9. Mass spectrometry reveals the assembly pathway of encapsulated ferritins and highlights a dynamic ferroxidase interface.
- Author
-
Ross, Jennifer, Lambert, Thomas, Piergentili, Cecilia, He, Didi, Waldron, Kevin J., Mackay, C. Logan, Marles-Wright, Jon, and Clarke, David J.
- Subjects
TANDEM mass spectrometry ,MASS spectrometry - Abstract
Encapsulated ferritins (EncFtn) are a recently characterised member of the ferritin superfamily. EncFtn proteins are sequestered within encapsulin nanocompartments and form a unique biological iron storage system. Here, we use native mass spectrometry and hydrogen–deuterium exchange mass spectrometry to elucidate the metal-mediated assembly pathway of EncFtn. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
10. Interactive vanKrevelen diagrams -- Advanced visualisation of mass spectrometry data of complex mixtures.
- Author
-
Kew, William, Blackburn, John W.T., Clarke, David J., and Uhrín, Dušan
- Subjects
MASS spectrometry ,SPECTROMETRY - Published
- 2017
- Full Text
- View/download PDF
11. Mass spectrometry analysis of the oxidation states of the pro-oncogenic protein anterior gradient-2 reveals covalent dimerization via an intermolecular disulphide bond.
- Author
-
Clarke, David J., Murray, Euan, Faktor, Jakub, Mohtar, Aiman, Vojtesek, Borek, MacKay, C. Logan, Smith, Pat Langridge, and Hupp, Ted R.
- Subjects
- *
MASS spectrometry , *OXIDATION states , *ONCOGENIC proteins , *DIMERIZATION , *INTERMOLECULAR interactions - Abstract
Anterior Gradient-2 (AGR2) is a component of a pro-oncogenic signalling pathway that can promote p53 inhibition, metastatic cell migration, limb regeneration, and cancer drug-resistance. AGR2 is in the protein-disulphide isomerase superfamily containing a single cysteine (Cys-81) that forms covalent adducts with its client proteins. We have found that mutation of Cysteine-81 attenuates its biochemical activity in its sequence-specific peptide docking function, reduces binding to Reptin, and reduces its stability in cells. As such, we evaluated how chemical oxidation of its cysteine affects its biochemical properties. Recombinant AGR2 spontaneously forms covalent dimers in the absence of reductant whilst DTT promotes dimer to monomer conversion. Mutation of Cysteine-81 to alanine prevents peroxide catalysed dimerization of AGR2 in vitro , suggesting a reactive cysteine is central to covalent dimer formation. Both biochemical assays and ESI mass spectrometry were used to demonstrate that low levels of a chemical oxidant promote an intermolecular disulphide bond through formation of a labile sulfenic acid intermediate. However, higher levels of oxidant promote sulfinic or sulfonic acid formation thus preventing covalent dimerization of AGR2. These data together identify the single cysteine of AGR2 as an oxidant responsive moiety that regulates its propensity for oxidation and its monomeric-dimeric state. This has implications for redox regulation of the pro-oncogenic functions of AGR2 protein in cancer cells. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
12. Probing the Conformational Diversity of Cancer-Associated Mutations in p53 with Ion-Mobility Mass Spectrometry.
- Author
-
Jurneczko, Ewa, Cruickshank, Faye, Porrini, Massimiliano, Clarke, David J., Campuzano, Iain D. G., Morris, Michael, Nikolova, Penka V., and Barran, Perdita E.
- Published
- 2013
- Full Text
- View/download PDF
13. Top-down protein sequencing by CID and ECD using desorption electrospray ionisation (DESI) and high-field FTICR mass spectrometry
- Author
-
Stokes, Adam A., Clarke, David J., Weidt, Stefan, Langridge-Smith, Pat, and Mackay, C. Logan
- Subjects
- *
AMINO acid sequence , *DISSOCIATION (Chemistry) , *MASS spectrometry , *CIRCULAR dichroism , *ELECTROSPRAY ionization mass spectrometry , *MYOGLOBIN , *CYTOCHROME c , *ION cyclotron resonance spectrometry - Abstract
Abstract: We report high resolution spectra for the medium molecular weight proteins myoglobin and cytochrome-c obtained using a custom desorption electrospray ionisation (DESI) source coupled to a Bruker Daltonics 12T Apex Qe Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS). The DESI source was designed for accurate alignment and reproduction of critical geometric variables. A two axis motorised stage was included to enable automated rastering of the sample under the DESI plume. Spectra for the intact proteins have been obtained under single-acquisition conditions and a top-down analysis of cytochrome-c was performed using both collision induced dissociation (CID) and electron capture dissociation (ECD) of the isolated [M+15H]15+ charge state. The sequence coverage is comparable to that obtained using electrospray ionisation, demonstrating the utility of top-down protein analysis by DESI FTICR-MS. [Copyright &y& Elsevier]
- Published
- 2010
- Full Text
- View/download PDF
14. Interrogating the Molecular Details of the Peroxiredoxin Activity of the Escherichia coli Bacterioferritin Comigratory Protein Using High-Resolution Mass Spectrometryt.
- Author
-
Clarke, David J., Mackay, C. Logan, Campopiano, Dominic J., Langridge-Smith, Pat, and Brown, Alan R.
- Subjects
- *
ESCHERICHIA coli , *PEROXIDASE , *MASS spectrometry , *ION cyclotron resonance spectrometry , *HYDROGEN peroxide , *SULFENIC acids - Abstract
Bacterioferritin comigratory protein (BCP) is a bacterial thioredoxin-dependent thiol peroxidase that reduces a variety of peroxide substrates. Using high-resolution Fourier transform ion cyclotron resonance mass spectrometry coupled with top-down fragmentation techniques, we have analyzed the mechanistic details of hydrogen peroxide reduction by E. co/i BCP. We show here that catalysis occurs via an atypical two-cysteine peroxiredoxin pathway. A transient sulfenic acid is initially formed on Cys-45, before resolution by the formation of an intramolecular disulfide bond between Cys-45 and Cys-50. This oxidized BCP intermediate is shown to be a substrate for reduction by thioredoxin, completing the catalytic cycle. Although we invoke Cys-50 in the catalytic cycle of Escherichia co/i bacterioferritin comigratory protein (BCP), a previous study had shown that this residue was not absolutely required for peroxiredoxin activity. In order to explain these apparently conflicting phenomena, we analyzed the reaction of a C5OS BCP mutant with peroxide. We show that this mutant BCP enzyme adopts a different and novel mechanistic pathway. The C5OS BCP mutant reacts with peroxide to form a sulfenic acid on Cys-45, in the same manner as wild-type BCP. However, the nascent intermediate is then resolved by reaction with Cys-45 from a second BCP molecule, resulting in a dimeric intermediate containing an intermolecular disulfide bond. We further show that this novel resolving complex is a substrate for reduction by thioredoxin. The importance of our results in furthering the understanding of catalysis, within BCP family is discussed. [ABSTRACT FROM AUTHOR]
- Published
- 2009
- Full Text
- View/download PDF
15. An affinity purification procedure to isolate oxidized p53
- Author
-
Scotcher, Jenna, Clarke, David J., and Langridge-Smith, Pat R.R.
- Subjects
- *
P53 protein , *OXIDATION-reduction reaction , *CHROMATOGRAPHIC analysis , *TUMOR suppressor proteins , *BIOTIN , *HOMOGENEITY - Abstract
Abstract: Oxidation of cysteine is now known to serve as a fundamental mechanism to control protein function or activity. Many redox-regulated proteins do not oxidize to homogeneity, resulting in a mixture of reduced and oxidized species which cannot be separated chromatographically. Here we describe a protocol for the separation of reduced and oxidized forms of the tumor suppressor protein p53. This purification method relies on the reversible labeling of thiol groups with biotin and exploitation of the ultrastrong biotin–avidin interaction. This purification procedure can be applied to other cysteine-containing proteins where enrichment of the oxidized form is required. [Copyright &y& Elsevier]
- Published
- 2012
- Full Text
- View/download PDF
16. Preparation of isotopically labelled recombinant β-defensin for NMR studies
- Author
-
Seo, Emily S., Vargues, Thomas, Clarke, David J., Uhrín, Dušan, and Campopiano, Dominic J.
- Subjects
- *
RECOMBINANT proteins , *PEPTIDE antibiotics , *RADIOLABELING , *ANTI-infective agents , *CHEMOKINES , *MOLECULAR structure , *X-ray crystallography , *NUCLEAR magnetic resonance spectroscopy - Abstract
Abstract: β-Defensins are a family of cationic peptides that contain six invariant cysteine residues that form characteristic disulfide bonds between Cys1–Cys5, Cys2–Cys4 and Cys3–Cys6. They have been shown to act as potent antimicrobial agents and chemokines. Human β-defensin 2 (HBD2) was first isolated from psoriatic skin lesions and the structure of this peptide has been solved by X-ray crystallography and NMR spectroscopy both of which are consistent with a fold that contains an N-terminal α-helix and three antiparallel β-strands. Here, we report the expression and purification of the first isotopically labelled β-defensin (15N HBD2) with 100% incorporation of 15N using a recombinant Escherichia coli method. Multidimensional NMR spectroscopy experiments: 2D 1H–15N HSQC, 3D HSQC-TOCSY and 3D HSQC-NOESY allows for the assignment of resonances with no overlapping or ambiguous peaks. This isotopically labelled peptide is highly suitable for studying the interactions between HBD2 and a range of components from both the mammalian immune system and bacterial pathogens. [Copyright &y& Elsevier]
- Published
- 2009
- Full Text
- View/download PDF
17. Is it biologically relevant to measure the structures of small peptides in the gas-phase?
- Author
-
Barran, Perdita E., Polfer, Nick C., Campopiano, Dominic J., Clarke, David J., Langridge-Smith, Patrick R.R., Langley, Ross J., Govan, John R.W., Maxwell, Alison, Dorin, Julia R., Millar, Robert P., and Bowers, Michael T.
- Subjects
- *
LUTEINIZING hormone releasing hormone , *MASS spectrometry , *DISSOCIATION (Chemistry) , *SCISSION (Chemistry) - Abstract
Abstract: Recent developments in sample introduction of biologically relevant molecules have heralded a new era for gas-phase methods of structural determination. One of the biggest challenges is to relate gas-phase structures, often measured in the absence of water and counter ions, with in vivo biologically active structures. An advantage of gas-phase based techniques is that a given peptide can be analysed in a variety of different forms, for example, as a function of charge state, or with additional water molecules. Molecular modelling can provide insight into experimental findings and help elucidate the differences between structural forms. Combining experiment and theory provides a thorough interrogation of candidate conformations. Here two important naturally occurring peptide systems have been examined in detail and results are assessed in terms of their biological significance. The first of these is gonadotropin-releasing hormone (GnRH), a decapeptide which is the central regulator of the reproductive system in vertebrates. We have examined several naturally occurring variants of this peptide using Ion Mobility Mass Spectrometry and Electron Capture Dissociation (ECD) in conjunction with Fourier Transform Ion Cyclotron Mass Spectrometry (FT-ICR-MS). Candidate conformations are modelled using the AMBER force field. Single amino acid changes, for example Gly6→Ala6, or Ala6→D-Ala6, have observable effects on the gas phase structure of GnRH. It has been shown that evolutionary primary sequence variations are key to the biological activity of GnRH, and it is thought that this is due to different binding affinities at target receptors. This work provides strong evidence that this activity is structurally based. The second system examined is the relationship between the quaternary structure and activity of two novel β-defensins. FT-ICR mass spectrometry has been employed to characterize di-sulphide bridging and dissociation based experiments utilised to investigate their structural core. Defr1, with five cysteines, exists as a covalently bound disulphide linked dimer; Defr1 Y5C with six cysteines also is observed as a dimer, but non-covalently bound, suggesting that this defensin has a tendency to aggregate. The activity of Defr1 is 10 times higher than that of Defr1 Y5C when tested against the pathogen Pseudomonas aeruginosa. The results from these studies could inform future design of novel GnRH type ligands and anti-microbial agents, and illustrate the power of gas-phase based techniques for solving peptide structures. [Copyright &y& Elsevier]
- Published
- 2005
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.