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Mass Spectrometric Study of Protein and Protein Ligand Complexes
Başlık:
Mass Spectrometric Study of Protein and Protein Ligand Complexes
Yazar:
Ju, Yue, author.
ISBN:
9780438096820
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (177 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 79-11(E), Section: B.
Advisors: Vicki Wysocki.
Özet:
Application of mass spectrometry (MS) to study quaternary structures of macromolecules and how they interact with the binding partners is of growing interest in the structural biology field. The research presented in this dissertation has three aims: (1) applying MS for protein detection, (2) applying MS for the study of protein-protein and protein-ligand complexes, and (3) developing a novel ionization method for protein complexes for potential clinical applications. One way to approach the protein detection application is to selectively capture target proteins from complex biological samples and subsequently identify them online in a matrix assisted laser desorption/ionization time-of-flight mass spectrometer based on their unique m/z. For sequence characterization, enzymatically digested peptides from captured proteins are subjected to tandem MS (MS/MS) to map the identities of captured proteins. This approach is employed to develop a membrane receptor embedded affinity capture platform coupled with MS for bacterial toxin detection (Chapter 2). The challenge of working with membrane receptors for immobilization onto the capture surface can be overcome by polymerization of the planar supported lipid bilayer for the reconstitution. Another way to reconstitute membrane receptors is to use nanodiscs, which are utilized in Chapter 3 to solubilize ganglioside GM1 for pentameric cholera toxin B (CTB) binding. MS/MS can be used to dissociate protein-protein or protein-ligand complexes into smaller sub-complexes for structure elucidation. A commonly used gas phase dissociation method, collision induced dissociation (CID), usually is not sufficient in providing informative dissociation pathways reflective of the native quaternary structures for protein-protein and protein-ligand complexes because it causes prevalent unfolding. An alternative MS/MS method, surface induced dissociation (SID), has proven to be powerful in revealing quaternary structures for large protein complexes. In chapter 3, SID MS/MS is applied to dissociate GM1 bound CTB complex. Guide by the crystal structure, the feasibility of SID MS/MS for examining ligand binding regions in protein-ligand complexes is revealed. The GM1 ligand which contacts two adjacent protein subunits with unequal affinity is retained on either of the subunits with different probabilities after SID of the GM1 bound CTB complex into sub-complexes. This implies the potential application of SID MS/MS for the study of ligand binding in protein complexes. The roles of inter- and intra-subunit structures in two pentameric protein complexes with known crystal structures in determining their SID MS/MS dissociation pathways are also examined. The application of MS in structure elucidation of protein-protein and protein-ligand complexes is extended to structure elucidation of the membrane protein complexes (chapter 5) and protein-oligonucleotide binding (chapter 6) in this work. Paper spray (PS) ionization for studying protein complexes is approached as an endeavor for fundamental ionization method study for protein complexes. It is proved that with optimized instrumental and experimental parameters, native-like non-covalent protein complexes can be successfully ionized by PS showing that this method is promising in analyzing protein complexes in complex biological samples.
Notlar:
School code: 0168
Konu Başlığı:
Tüzel Kişi Ek Girişi:
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(696372.1) | 696372-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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