Characterization and quantitation of protein adducts using mass spectrometry

Persistent Link:
http://hdl.handle.net/10150/280025
Title:
Characterization and quantitation of protein adducts using mass spectrometry
Author:
Mason, Daniel
Issue Date:
2002
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:
Protein modifications by reactive intermediates may have a causal role in cellular toxicity but information about which proteins are modified is limited. In order to address identification of protein targets a novel method for detection of adducted peptides based on adduct-specific fragmentations in tandem mass spectrometry (MS/MS) was developed. This method consists of characterizing the MS/MS fragmentation of adducted-model peptides to identify adduct specific features and screening MS/MS spectra for characteristic features of adduction. Benzoquinone (BZQ) and glutathione-conjugated benzoquinone (GS-BZQ) were selected as model electrophiles to develop this method and adducts were prepared with model peptides to identify characteristic features of adduct fragmentation in MS/MS experiments. BZQ-adducted peptides fragmented to give adduct-derived fragments of ion pairs of 141/142 and 211 and a neutral loss of 142. GS-BZQ-adducted peptides fragmented to give adduct-derived fragments of neutral losses of 74, 129, 273, and 447, charged losses of 274 and 448 and ion pairs of 515 and 129. We suggest that these adduct-specific fragments can be used to detect adducted peptides. Subsequently, the data reduction algorithm SALSA was developed to screen MS/MS spectra for spectral characteristics including neutral losses, charged losses and ion pairs in order to facilitate adduct detection. SALSA scores multiple types of spectral features simultaneously and reports a combined score for each spectrum, and search criteria can be arranged in a hierarchal manner for more selective searching. The SALSA algorithm was used to screen spectra of a BSA digest treated with GS-BZQ for fragment characteristics of GS-BZQ-adduction, and spectra from six GS-BZQ modified peptides were ranked among the top twenty highest scoring spectra. Detection of unanticipated peptide modifications is illustrated using the motif-searching algorithm of SALSA which is described and searches for patterns of product ions without regard to precursor m/ z or position along the m/z axis. Finally, because the effects of adduction may depend on its abundance in the cell, a new stable isotope label for differential quantitation of peptide adducts is described. Relative quantitation using the label is linear across a 10,000 fold range of concentration ratios, standard deviation is less than 20%, and quantitation of multiple peptides in a BSA digest is reported. Styrene oxide adducts of hemoglobin are differentially quantified using the label and a concentration/adduct curve for the formation of eight peptide adducts is plotted.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Biology, Cell.; Health Sciences, Toxicology.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Pharmacology and Toxicology
Degree Grantor:
University of Arizona
Advisor:
Liebler, Daniel C.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleCharacterization and quantitation of protein adducts using mass spectrometryen_US
dc.creatorMason, Danielen_US
dc.contributor.authorMason, Danielen_US
dc.date.issued2002en_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.abstractProtein modifications by reactive intermediates may have a causal role in cellular toxicity but information about which proteins are modified is limited. In order to address identification of protein targets a novel method for detection of adducted peptides based on adduct-specific fragmentations in tandem mass spectrometry (MS/MS) was developed. This method consists of characterizing the MS/MS fragmentation of adducted-model peptides to identify adduct specific features and screening MS/MS spectra for characteristic features of adduction. Benzoquinone (BZQ) and glutathione-conjugated benzoquinone (GS-BZQ) were selected as model electrophiles to develop this method and adducts were prepared with model peptides to identify characteristic features of adduct fragmentation in MS/MS experiments. BZQ-adducted peptides fragmented to give adduct-derived fragments of ion pairs of 141/142 and 211 and a neutral loss of 142. GS-BZQ-adducted peptides fragmented to give adduct-derived fragments of neutral losses of 74, 129, 273, and 447, charged losses of 274 and 448 and ion pairs of 515 and 129. We suggest that these adduct-specific fragments can be used to detect adducted peptides. Subsequently, the data reduction algorithm SALSA was developed to screen MS/MS spectra for spectral characteristics including neutral losses, charged losses and ion pairs in order to facilitate adduct detection. SALSA scores multiple types of spectral features simultaneously and reports a combined score for each spectrum, and search criteria can be arranged in a hierarchal manner for more selective searching. The SALSA algorithm was used to screen spectra of a BSA digest treated with GS-BZQ for fragment characteristics of GS-BZQ-adduction, and spectra from six GS-BZQ modified peptides were ranked among the top twenty highest scoring spectra. Detection of unanticipated peptide modifications is illustrated using the motif-searching algorithm of SALSA which is described and searches for patterns of product ions without regard to precursor m/ z or position along the m/z axis. Finally, because the effects of adduction may depend on its abundance in the cell, a new stable isotope label for differential quantitation of peptide adducts is described. Relative quantitation using the label is linear across a 10,000 fold range of concentration ratios, standard deviation is less than 20%, and quantitation of multiple peptides in a BSA digest is reported. Styrene oxide adducts of hemoglobin are differentially quantified using the label and a concentration/adduct curve for the formation of eight peptide adducts is plotted.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectBiology, Cell.en_US
dc.subjectHealth Sciences, Toxicology.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplinePharmacology and Toxicologyen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorLiebler, Daniel C.en_US
dc.identifier.proquest3053892en_US
dc.identifier.bibrecord.b4281313xen_US
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