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Hydrogen-deuterium exchange studies ...

Rojsajjakul, Teerapat.

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Hydrogen-deuterium exchange studies by mass spectrometry: A method for obtaining conformational structures of proteins in solution.

紀錄類型:	書目-語言資料,印刷品 : Monograph/item
正題名/作者:	Hydrogen-deuterium exchange studies by mass spectrometry: A method for obtaining conformational structures of proteins in solution./
作者:	Rojsajjakul, Teerapat.
面頁冊數:	149 p.
附註:	Source: Dissertation Abstracts International, Volume: 75-08(E), Section: B.
Contained By:	Dissertation Abstracts International75-08B(E).
標題:	Chemistry, Analytical. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3618139
ISBN:	9781303862274

Hydrogen-deuterium exchange studies by mass spectrometry: A method for obtaining conformational structures of proteins in solution.
Rojsajjakul, Teerapat.

Hydrogen-deuterium exchange studies by mass spectrometry: A method for obtaining conformational structures of proteins in solution. - 149 p.

Source: Dissertation Abstracts International, Volume: 75-08(E), Section: B.

Thesis (Ph.D.)--West Virginia University, 2014.

Hydrogen-deuterium exchange (HDX) coupled with electrospray ionization (ESI) in mass spectrometry (MS) has become an important method in the study of protein structure and dynamics. It provides an opportunity for obtaining conformational structures of protein motions in physiological conditions and offers unparalleled limits of detection. This method relies on the fact that the accessibility of exchangeable amide hydrogens of a protein to D2O induces H/D isotopic exchange. The exchangeable amide hydrogens that lack hydrogen bonding when exposed to solvent are exchanged rapidly. Protected amide hydrogens from tightly folded elements and have hydrogen bonding exchange slow and can be detected by mass spectrometry. This dissertation discusses basic fundamentals of both the conventional HDX ESI MS method that is called bottom-up approach that utilizes an enzymatic digestion and gas phase fragmentation method or a top-down approach to obtain hydrogen isotopic exchange at specific amide hydrogens.

ISBN: 9781303862274Subjects--Topical Terms:

586156
Chemistry, Analytical.

Hydrogen-deuterium exchange studies by mass spectrometry: A method for obtaining conformational structures of proteins in solution.
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245 1 0 $a Hydrogen-deuterium exchange studies by mass spectrometry: A method for obtaining conformational structures of proteins in solution.
300 $a 149 p.
500 $a Source: Dissertation Abstracts International, Volume: 75-08(E), Section: B.
500 $a Adviser: Fred L. King.
502 $a Thesis (Ph.D.)--West Virginia University, 2014.
520 $a Hydrogen-deuterium exchange (HDX) coupled with electrospray ionization (ESI) in mass spectrometry (MS) has become an important method in the study of protein structure and dynamics. It provides an opportunity for obtaining conformational structures of protein motions in physiological conditions and offers unparalleled limits of detection. This method relies on the fact that the accessibility of exchangeable amide hydrogens of a protein to D2O induces H/D isotopic exchange. The exchangeable amide hydrogens that lack hydrogen bonding when exposed to solvent are exchanged rapidly. Protected amide hydrogens from tightly folded elements and have hydrogen bonding exchange slow and can be detected by mass spectrometry. This dissertation discusses basic fundamentals of both the conventional HDX ESI MS method that is called bottom-up approach that utilizes an enzymatic digestion and gas phase fragmentation method or a top-down approach to obtain hydrogen isotopic exchange at specific amide hydrogens.
520 $a Insulin, substance P and ubiquitin were used as model peptide and proteins to investigate the utility of these approaches. Bovine insulin was used to study the reduction of disulfide bonds using TECP as a reducing agent after the hydrogen isotopic reaction. The results show that the peptic fragments obtained from this method are smaller than those from the previous study without the reduction of disulfide bonds. Therefore, this approach offers more structural details and ease in identifying the peptic fragments. However, the drawback from back exchange reaction during the reduction time and chromatographic separation requires further study. Substance P was used to investigate gas phase fragmentation of the deuterated peptide by electron capture dissociation (ECD). The results show that this top-down approach is feasible for HDX MS method. The ECD fragmentation of the doubly charged ion of deuterium labeled substance P after chromatographic separation method that eliminated the rapid exchanged deuteriums at the side chains and termini produces less scrambling. Ubiquitin was used to further investigate gas phase fragmentation of deuterium labeled protein by ECD. The surprising results from direct infusion show that the higher charge states gain more deuteriums than the lower ones. On the contrary, the gas phase fragmentation of the deuterated protein from chromatographic separation that eliminated the rapid exchanged deuteriums show the same number of deuteriums for all charge states. Thus, the results from both experiments provide the labile exchanged deuterium locations. Human insulin was used to study protein conformational change in the presence of Congo red (CR) by pulsed HDX ESI MS. CR is known as an inhibitor of insulin fibrillization by binding at the C-terminus of the B-Chain of insulin. Remarkably, the ESI mass spectrum of labeled protein in the presence of CR shows bimodal pattern, suggesting that CR binds with the partial unfolded intermediate structure of the protein. Moreover, the local HDX resulted from peptic digestion show that the conformational structure of the insulin in the presence of CR is different from that of the native form.
520 $a Overall, the results presented highlight the ability of the methods to provide insight into the conformational changes of proteins in solution.
590 $a School code: 0256.
650 4 $a Chemistry, Analytical. $3 586156
650 4 $a Chemistry, Biochemistry. $3 1017722
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710 2 $a West Virginia University. $b Eberly College of Arts & Sciences. $3 2099039
773 0 $t Dissertation Abstracts International $g 75-08B(E).
790 $a 0256
791 $a Ph.D.
792 $a 2014
793 $a English
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