Graduate Thesis Or Dissertation


Mass-Spectrometric Methods for OxPTM Characterization and Quantitative Proteomics of Oxidative Stress Response Public Deposited

Downloadable Content

Download PDF


Attribute NameValues
  • Oxidative stress is defined as an imbalance that favors the production of reactive oxygen species (ROS) over an organism's antioxidant defense. ROS have the ability to damage, either directly or indirectly, biomolecules including DNA, proteins, carbohydrates, and lipids. Various pathological conditions and environmental and chronic diseases have been associated with elevated levels of oxidative stress. Protein carbonyls have been widely recognized as markers of oxidative damage to proteins. Protein carbonylation can occur by direct modifications of ROS and metal-catalyzed oxidation (MCO) of specific amino acid residues. Another source of generating carbonyl groups on proteins is the covalent modification of amino acid side chains by reactive lipid peroxidation products (LPPs), a large variety of which are produced during oxidation of polyunsaturated fatty acids (PUFAs). Many LPPs are electrophiles and thus readily react with nucleophilic groups in proteins. In this thesis, I describe the development, application and evaluation of contemporary mass spectrometric methods for the detection, characterization, and quantification of protein modifications associated with the conditions of elevated levels of oxidative stress and for profiling responses of the proteome to oxidative stress in the context of exposure to ionizing radiation with focus on mouse hippocampus, a brain region important for learning and memory. Firstly, we explored the applicability of travelling wave ion mobility mass spectrometry (TWIM-MS) in conjunction with collision-induced dissociation (CID) for characterizing protein modifications caused by reactive lipid peroxidation products with focus on a) 4-hydroxy-nonenal (HNE), an alpha, beta-unsaturated hydroxyalkenal, and b) levuglandins, a group of isomeric γ-ketoaldehydes. We tested the capabilities of TWIM-MS in combination with collision-induced dissociation for the analysis of positional isomers of peptides modified by 4-hydroxy-2-nonenal (HNE). The model peptides that we utilized for these experiments were derived from glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme of glycolysis and a known target of HNE. We were able to demonstrate that TWIM-MS can separate positional isomers of peptides with a HNE modification on histidine residues at different sequence positions by differentiating them according to drift time distributions (DTDs). Similarly, synthetic peptides, which had the same sequence as the first set of model peptides but the histidine residues were replaced by cysteine residues, were well resolved by TWIM-MS in the gas phase. The combination of ion mobility with CID resulted in product ion spectra drift-time aligned with the precursor ions and allowed the positional differentiation of the HNE adduction despite neutral loss of the modification during collision-induced dissociation. Extracted drift time chromatograms of distinct fragment ions provided a visualization tool that was beneficial for the site-specific assignments of HNE adduction. The combination of TWIM-MS with CID provides another opportunity beyond conventional tandem mass spectrometric methods for the analysis of positional isomers of peptides with PTMs. Although this study focused on peptide-HNE adducts, we expect that TWIM-MS is applicable to the analysis of other PTMs as well and advances our capability of obtaining site-specific information for PTMs. TWIM-MS was also explored for the analysis of levuglandins/isoketals (or γ-ketoaldehydes) lysine adducts. Non-enzymatic oxidation and cyclization of arachidonic acid (AA) leads to the formation of four regio-isomers of γ-ketoaldehydes. The γ-ketoaldehyde functionality is highly reactive toward primary amino groups in biomolecules. In proteins, modification by γ-ketoaldehydes leads to formation of lactam adducts. The development of a fast and reliable method is reported for the analysis and separation of four regio-isomeric isoketal-lysyl-lactam adducts by utilizing ultra performance liquid chromatography (UPLC) in conjunction with TWIM-MS. This novel strategy is potentially extendable toward the detection, characterization and quantification of isoketal-lactam adducts in pre-clinical studies, and ultimately possibly applicable in disease diagnosis and drug developmental efforts that target pathological conditions associated with oxidative stress. Secondly, a high resolution LC-MS/MS-based methodology was developed for quantification of α-amino adipic semialdehyde () in biological samples. Clinical and public health awareness of ionizing radiation (IR) has increased the number of studies concerned with injury and impact of IR on biological systems. Oxidative stress is one of the major injuries resulting from IR. and the development of a reliable and robust biodosimetry method for assessing the injury of irradiation at the level of oxidative stress is highly needed. Protein carbonyls widely recognized as a biomarker of oxidative stress under various pathological conditions. However, due to the chemical diversity of protein carbonyls and their low concentrations, an analytical strategy was choosen that is based on using α-AAS as marker of protein carbonylation and oxidative insult after total proteolysis.. A strategy was developed for the quantitation of α-AAS that combines a high sensitivity LC-ESI-MS/MS MRM[superscript HR] approach and a pronase-based hydrolysis protocol. In addition, a novel derivatization reagent was utilized that features a permanently charged, quaternary aminooxy (QAO) reactive group that targets the aldehyde/keto functionality group The introduction of a permanent charge into the analyte enhances the ESI-MS/MS sensitivity for detection. The utilization of pronase allows mild hydrolysis and minimizes the artificial formation of oxidatively modified amino acids. The derivatization method was first applied as a "proof of concept" for quantitation of α-AAS in plasma samples after metal catalyzed oxidation (MCO). , The protocol was then extended to determine α-AAS levels in hippocampal tissues of individual mice that were exposed to low dose (1Gy) irradiation The extension of the protocol to a multiplex MRM[superscript HR] assay was allowed determining level estimates of three carbonylated amino acids, namely α-AAS,γ -glutamic semialdehyde and N-formyl kynurenine, semi-quantitatively in a single analytical run In summary, the methodology introduced here provides a sensitive, reliable and robust approach to quantify carbonylated amino acids without any enrichment, and greatly improves the measurement efficiency of carbonylated amino acids as markers of oxidative insult in complex biological samples... Thirdly, we applied and evaluated a label-free LC-IM-MS[superscript E] quantification method for determining proteome changes of the mouse hippocampus to low dose irradiation at 1 Gy. In the present study, 5.5-month-old male C57BL/6J mice underwent contextual fear conditioning training, were subsequent irradiated with low-dose whole body ionizing radiation (X-rays, 1 Gy) and then tested for hippocampus-dependent contextual fear memory 24 h post-irradiation. Under these experimental conditions, there was no significant effect of irradiation on contextual memory. We hypothesized that compensatory radiation-induced changes in the hippocampus might have masked cognitive injury. Analysis of the observed proteome changes using biological pathway enrichment tools suggested a shift in mitochondrial energy metabolism, down-regulation of myelination and up-regulation of calcium-dependent synaptic plasticity. The proteome alterations were consistent with elevated long-term potentiation (LTP) and decreased long-term depression (LTD) suggesting improved synaptic transmission and an enhanced efficiency of neurotransmitter release, which would be consistent with the observed comparable contextual fear memory performance of the mice following post-training whole body or sham-irradiation.
Resource Type
Date Available
Date Issued
Degree Level
Degree Name
Degree Field
Degree Grantor
Commencement Year
Committee Member
Academic Affiliation
Non-Academic Affiliation
Rights Statement
Peer Reviewed



This work has no parents.

In Collection: