Persistent Link:
http://hdl.handle.net/10150/289989
Title:
Protein targets and metabolites of 1,1-dichloroethylene
Author:
Jones, Juliet Ann
Issue Date:
2001
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Xenobiotics and their metabolites are capable of covalent modification of proteins that may lead to cellular injury. Determining the identity of modified proteins, however, has to date been hindered by available analytical instrumentation. Advances in mass spectrometry now allow for routine analysis of large biomolecules. This has given rise to the field of proteomics , which uses mass spectrometry in conjunction with various separation techniques to gain information about large numbers of proteins. This dissertation describes the development of a new proteomic methodology to identify unknown protein targets and its application to identifying protein targets and metabolites of 1,1-dichloroethylene (1,1-DCE). 1,1-DCE undergoes P450 bioactivation in hepatocytes to produce 1,1-DCE oxide and 2-chloroacetyl chloride. These either modify proteins directly, or form the glutathione conjugate S-(2-chloroacetyl)glutathione, which in turn is capable of protein cysteinyl sulfhydryl alkylation. In animals, exposure to 1,1-DCE results in selective injury to the biliary canalicular membrane. Biochemical and physiological evidence suggests damage to the canalicular membrane transport proteins. Putative 1,1-DCE metabolite adducts, including S-carboxymethylated and 2-chloroacetylated peptides and GSCOCH₂-S-cys-peptide adducts, were synthesized using model peptides containing one or two cysteines. The adducts were analyzed by ESI-MS-MS and resulted in fragmentation patterns characteristic of the adduct moiety, including fragment ions, losses from the parent ion and pairs of ions separated by the mass of the modified cysteine residue. The data reduction algorithm SALSA was developed to search for user-specified fragmentation characteristics in MS-MS data dependent scans. Bile samples obtained from animals exposed to 1,1-DCE were analyzed by ESI-MS-MS. SALSA was then used to search the data for spectra containing adduct-specific fragmentation. Five hepatic S-carboxymethylated proteins were identified, as were the 1,1-DCE metabolites S-carboxymethylglutathione, S-(cysteinylacetyl)glutathione and the cyclic product of the intermolecular rearrangement of S-(2-chloroacetyl)glutathione. This work demonstrates the use of mass spectrometry to characterize unknown, modified proteins in complex mixtures without the use of radio- or immunochemical labeling.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Health Sciences, Toxicology.; Chemistry, Analytical.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Chemistry
Degree Grantor:
University of Arizona
Advisor:
Liebler, Daniel C.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleProtein targets and metabolites of 1,1-dichloroethyleneen_US
dc.creatorJones, Juliet Annen_US
dc.contributor.authorJones, Juliet Annen_US
dc.date.issued2001en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractXenobiotics and their metabolites are capable of covalent modification of proteins that may lead to cellular injury. Determining the identity of modified proteins, however, has to date been hindered by available analytical instrumentation. Advances in mass spectrometry now allow for routine analysis of large biomolecules. This has given rise to the field of proteomics , which uses mass spectrometry in conjunction with various separation techniques to gain information about large numbers of proteins. This dissertation describes the development of a new proteomic methodology to identify unknown protein targets and its application to identifying protein targets and metabolites of 1,1-dichloroethylene (1,1-DCE). 1,1-DCE undergoes P450 bioactivation in hepatocytes to produce 1,1-DCE oxide and 2-chloroacetyl chloride. These either modify proteins directly, or form the glutathione conjugate S-(2-chloroacetyl)glutathione, which in turn is capable of protein cysteinyl sulfhydryl alkylation. In animals, exposure to 1,1-DCE results in selective injury to the biliary canalicular membrane. Biochemical and physiological evidence suggests damage to the canalicular membrane transport proteins. Putative 1,1-DCE metabolite adducts, including S-carboxymethylated and 2-chloroacetylated peptides and GSCOCH₂-S-cys-peptide adducts, were synthesized using model peptides containing one or two cysteines. The adducts were analyzed by ESI-MS-MS and resulted in fragmentation patterns characteristic of the adduct moiety, including fragment ions, losses from the parent ion and pairs of ions separated by the mass of the modified cysteine residue. The data reduction algorithm SALSA was developed to search for user-specified fragmentation characteristics in MS-MS data dependent scans. Bile samples obtained from animals exposed to 1,1-DCE were analyzed by ESI-MS-MS. SALSA was then used to search the data for spectra containing adduct-specific fragmentation. Five hepatic S-carboxymethylated proteins were identified, as were the 1,1-DCE metabolites S-carboxymethylglutathione, S-(cysteinylacetyl)glutathione and the cyclic product of the intermolecular rearrangement of S-(2-chloroacetyl)glutathione. This work demonstrates the use of mass spectrometry to characterize unknown, modified proteins in complex mixtures without the use of radio- or immunochemical labeling.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectHealth Sciences, Toxicology.en_US
dc.subjectChemistry, Analytical.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineChemistryen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorLiebler, Daniel C.en_US
dc.identifier.proquest3010260en_US
dc.identifier.bibrecord.b41712225en_US
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