[PDF] Study Of Non Covalent Protein Carbohydrate Interactions Using Electrospray Ionization Mass Spectrometry eBook

Author : Sanaz Nikjah
Publisher :
Page : 171 pages
File Size : 41,65 MB
Release : 2015
Category : Biomolecules
ISBN :

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This thesis describes the application of electrospray ionization mass spectrometry (ESI-MS) techniques to identify and quantify antibody-antigen interactions in vitro. Also potential application of nanodiscs (ND) as membrane model in ESI-MS and surface plasmon resonance (SPR) spectroscopy has been investigated. A direct nanoelectrospray mass spectrometry assay (nanoESI-MS) was developed for quantification of specific binding between a monoclonal antibody (mAb) and its cognate antigen. A qualitative investigation was performed on cross-reactivity of antibodies. NanoESI-MS was also employed to investigate the effects of fatty acid chain lengths on its degree of incorporation into ND. The results can be utilized for synthesis purposes where biologically important carbohydrates require a proper carbon chain for incorporation into ND. ND technology was also employed to investigate protein-ligand interaction using SPR spectroscopy. The results open up further investigation on the effect of the position of ganglioside binding sites incorporated into ND and ability of their binding to the receptors in SPR.

Author : Ling Han
Publisher :
Page : 293 pages
File Size : 43,62 MB
Release : 2015
Category : Electrospray ionization mass spectrometry
ISBN :

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The interactions between water-soluble proteins and carbohydrates found on the surfaces of cells play important roles in many physiological and pathological cellular processes. Carbohydrates function as receptors for signaling, cellular recognition and adhesion, and pathogen infections. This thesis focuses on the development and application of electrospray ionization mass spectrometry (ESI-MS) methods to detect and quantify protein-carbohydrate interactions in vitro. In Chapter 2, the intrinsic affinities (per binding site) of the protruding domain dimer (P dimer, 69 kDa) of the human norovirus (NoV) strain VA387 to a panel of 47 soluble analogs of histo-blood group antigens (HBGAs) were quantified using the direct ESI-MS assay. Our results revealed that the P dimer exhibits a broad specificity for the HBGAs and bind, albeit weakly (intrinsic association constants (Ka,int) of 102 - 103 M-1), to all of the oligosaccharides tested. Overall, the A and B antigens exhibit stronger binding than the H and Lewis antigens. In addition, the affinities are also affected by the precursor chain type of HBGAs but not by the chain length. In Chapter 3, the applicability of the catch-and-release (CaR)-ESI-MS assay for screening carbohydrate libraries against large protein complexes was demonstrated for the first time. Libraries containing as many as 146 compounds were screened against NoV VA387 subviral P particle (24-mer, 865 kDa). Notably, the results of the screening experiments revealed NoV interactions with oligosaccharides with structures found in human milk and the cell wall of mycobacteria. The affinities of these newly discovered ligands are comparable to those of the HBGA receptors. In Chapters 4 and 6, the direct ESI-MS assay was combined with a competitive binding strategy in order to measure the affinities of protein-carbohydrate interactions that can't be directly quantified by ESI-MS. In Chapter 4, the affinities of the NoV P particle and virus-like particle (VLP, 180-mer, 10.5 MDa) to HBGA ligands were quantified using the proxy protein ESI-MS method, which utilizes competitive protein binding. The results revealed that HBGA ligands exhibit similar affinities for the P particle and P dimer whereas the HBGA affinities for the VLP are consistently higher than those measured for the P dimer, but within a factor of three. In Chapter 6, the proxy ligand ESI-MS assay, which is on the basis of competitive ligand binding, combined with nanodisc technology to solubilize glycolipids was used to determine the interactions of cholera toxin B subunit homopentamer (CTB5) with GM1, and a family 51 carbohydrate-binding module (CBM) with B type 2 tetrasaccharide neoglycolipid. A notable finding of this study is that the affinities of the glycolipid ligands in the nanodisc are lower, by a factor of ≤5, than those of the corresponding oligosaccharides in solution. In Chapter 5, the screening using CaR-ESI-MS assay revealed the first evidence that human NoVs bind to gangliosides. Moreover, affinities measurements were reported for the NoV VA387 P dimer, P particle and VLP, and VA115 P dimer for a series of ganglioside oligosaccharides. Notably, the ganglioside affinities are similar in magnitude to those of HBGA receptors for NoVs. Additional confirmation of NoV-ganglioside interactions was provided by the binding measurements using the enzyme-linked immunosorbent assays. Finally, also in Chapter 6, a systematic ESI-MS investigation aimed at elucidating the processes that influence binding of water-soluble proteins to glycolipids incorporated into nanodiscs was described. The interactions of CTB5 to GM1 nanodiscs studied by ESI-MS indicated that proteins bind reversibly to nanodisc-associated glycolipids, and that proteins possessing multiple ligand binding sites are able to interact with glycolipids originating from different nanodiscs. Moreover, the nature of the protein-glycolipid complexes detected by ESI-MS is likely to be influenced by the diffusion of glycolipids between nanodiscs.

Author : Yixuan Zhang
Publisher :
Page : 123 pages
File Size : 27,18 MB
Release : 2013
Category : Carbohydrates
ISBN :

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This thesis describes the development and application of electrospray ionization mass spectrometry (ESI-MS) based techniques to investigate protein-carbohydrate interactions in vitro. A catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay was developed for the identification of specific interactions between water-soluble multisubunit proteins and glycosphingolipids (GSL). The assay is of high sensitivity and specificity, and demonstrates the potential for discovering biologically relevant protein-GSL interactions. Collision-induced dissociation (CID) experiments and molecular dynamic simulations were performed to investigate the dissociation pathways of multisubunit protein-ligand complexes in the gas phase. The observation of multiple dissociation pathways suggests that collisional activation of multisubunit protein-ligand complexes in the gas phase is likely to induce significant changes to the nature of the protein-ligand interactions.

Author : Christoph A. Schalley
Publisher : John Wiley & Sons
Page : 593 pages
File Size : 20,8 MB
Release : 2009-09-08
Category : Science
ISBN : 0470131152

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Details the many benefits of applying mass spectrometry to supramolecular chemistry Except as a method for the most basic measurements, mass spectrometry (MS) has long been considered incompatible with supramolecular chemistry. Yet, with today's methods, the disconnect between these two fields is not warranted. Mass Spectrometry and Gas-Phase Chemistry of Non-Covalent Complexes provides a convincing look at how modern MS techniques offer supramolecular chemists a powerful investigatory toolset. Bringing the two fields together in an interdisciplinary manner, this reference details the many different topics associated with the study of non-covalent complexes in the gas phase. The text begins with brief introductions to supramolecular chemistry and such relevant mass spectrometric methods as ionization techniques, analyzers, and tandem MS experiments. The coverage continues with: How the analyte's transition into the gas phase changes covalent bonding How limitations and pitfalls in analytical methods may produce data misinterpretations Artificial supramolecular aggregates and their examination Biomolecules, their complexes, and their examination After the general remarks making up the first section of the book, the following sections describe specific experimental procedures and are illustrated with numerous examples and short tutorials. Detailed citations end each chapter. Mass spectrometrists, supramolecular chemists, students in these fields, and interested readers from other disciplines involving the study of non-covalent bonds will all value Mass Spectrometry and Gas-Phase Chemistry of Non-Covalent Complexes as an innovative and practical resource.