Identification, Characterization, and Quantification of Dicarbonyl Adducts in the Plasma Proteome in Type-2 Diabetes

Persistent Link:
http://hdl.handle.net/10150/145123
Title:
Identification, Characterization, and Quantification of Dicarbonyl Adducts in the Plasma Proteome in Type-2 Diabetes
Author:
Kimzey, Michael John
Issue Date:
2011
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:
Glyco-oxidation is linked to the pathophysiology of diabetes and diabetic complications. The process of glyco-oxidation generates reactive dicarbonyls, which form adducts on arginine residues in distributions throughout the proteome that are site-specific depending on the protein microenvironment. Dicarbonyl adducts are thus markers for glyco-oxidative stress. Various approaches using mass spectrometry permits the identification, localization, and quantification of these dicarbonyl adducts. Using MG as a model dicarbonyl, a shotgun proteomics approach identified the sites for modification of major plasma proteins. Thirty five sites on seven abundant plasma proteins were found, and investigation into the microenvironment surrounding the target arginine sites revealed a neighboring charged residue motif where adjacent residues were either negatively or positively charged. One of the sites identified was R257 in HSA, which is located in the important drug binding site I. We validated drug site I as a target for MG modification by the adaptation of two assays to monitor the effect of MG modification. MG significantly decreases the rate of hydrolysis of PGE2 in drug site I, and induces the displacement of prodan from drug site I. Molecular modeling of warfarin docking at drug site I with the MG-modified R257 resulted in significantly decreased binding and change in binding orientation. The oxidation products of susceptible residues methionine, tryptophan, and cysteine were evaluated using MRM of oxidized HSA peptides. Oxidation of methionine gave the M+16 single oxidized product, and M329 in HSA was the most responsive site. Oxidation of the sole W214 tryptophan produced the W+32 double oxidation product, and oxidation of C34 produced the C+48 triple oxidation product. MG, 3DG, and glucosone were evaluated for propensity to modify 12 HSA sites based on MRM of dicarbonyl modified HSA. Dicarbonyl modification was independent of arginine solvent accessibility. In a clinical study using nephropathy as an endpoint, sites of oxidation and modification of HSA by MG, 3DG, and glucosone were quantified by MRM. The most important variable among diabetic subjects was metformin use, and subjects taking metformin had significantly reduced markers for glyco-oxidation. These findings may be useful in the development of new diabetes therapies that aim to ameliorate glyco-oxidative stress.
Type:
Electronic Dissertation; text
Keywords:
diabetes; mass spectrometry; methylglyoxal; multiple reaction monitoring; oxidative stress; proteomics
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Pharmaceutical Sciences
Degree Grantor:
University of Arizona
Advisor:
Lau, Serrine S

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleIdentification, Characterization, and Quantification of Dicarbonyl Adducts in the Plasma Proteome in Type-2 Diabetesen_US
dc.creatorKimzey, Michael Johnen_US
dc.contributor.authorKimzey, Michael Johnen_US
dc.date.issued2011-
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.abstractGlyco-oxidation is linked to the pathophysiology of diabetes and diabetic complications. The process of glyco-oxidation generates reactive dicarbonyls, which form adducts on arginine residues in distributions throughout the proteome that are site-specific depending on the protein microenvironment. Dicarbonyl adducts are thus markers for glyco-oxidative stress. Various approaches using mass spectrometry permits the identification, localization, and quantification of these dicarbonyl adducts. Using MG as a model dicarbonyl, a shotgun proteomics approach identified the sites for modification of major plasma proteins. Thirty five sites on seven abundant plasma proteins were found, and investigation into the microenvironment surrounding the target arginine sites revealed a neighboring charged residue motif where adjacent residues were either negatively or positively charged. One of the sites identified was R257 in HSA, which is located in the important drug binding site I. We validated drug site I as a target for MG modification by the adaptation of two assays to monitor the effect of MG modification. MG significantly decreases the rate of hydrolysis of PGE2 in drug site I, and induces the displacement of prodan from drug site I. Molecular modeling of warfarin docking at drug site I with the MG-modified R257 resulted in significantly decreased binding and change in binding orientation. The oxidation products of susceptible residues methionine, tryptophan, and cysteine were evaluated using MRM of oxidized HSA peptides. Oxidation of methionine gave the M+16 single oxidized product, and M329 in HSA was the most responsive site. Oxidation of the sole W214 tryptophan produced the W+32 double oxidation product, and oxidation of C34 produced the C+48 triple oxidation product. MG, 3DG, and glucosone were evaluated for propensity to modify 12 HSA sites based on MRM of dicarbonyl modified HSA. Dicarbonyl modification was independent of arginine solvent accessibility. In a clinical study using nephropathy as an endpoint, sites of oxidation and modification of HSA by MG, 3DG, and glucosone were quantified by MRM. The most important variable among diabetic subjects was metformin use, and subjects taking metformin had significantly reduced markers for glyco-oxidation. These findings may be useful in the development of new diabetes therapies that aim to ameliorate glyco-oxidative stress.en_US
dc.typeElectronic Dissertationen_US
dc.typetexten_US
dc.subjectdiabetesen_US
dc.subjectmass spectrometryen_US
dc.subjectmethylglyoxalen_US
dc.subjectmultiple reaction monitoringen_US
dc.subjectoxidative stressen_US
dc.subjectproteomicsen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplinePharmaceutical Sciencesen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorLau, Serrine Sen_US
dc.contributor.committeememberWondrak, Georgen_US
dc.contributor.committeememberStump, Craigen_US
dc.contributor.committeememberTsaprailis, Georgeen_US
dc.contributor.committeememberMonks, Terrenceen_US
dc.contributor.committeememberLau, Serrine Sen_US
dc.identifier.proquest11531-
dc.identifier.oclc752261395-
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