Mass Spectrometry-Based High Throughput Approach for Identification of Molecular Modification of Oxidative Process in Respiratory Diseases
Date of Award
Oxidation, Eosinophils, Tyrosine, Respiratory organs -- Diseases, Mass spectrometry, GC/MS, LC-MS/MS, Eosinophil, Oxidation, Bromotyrosine, Tyrosine
Eosinophil peroxidase (EPO) and myeloperoxidase (MPO) have been implicated in generating reactive species and promoting oxidative modifications in numerous diseases. The detection and identification of potential pathways has been proven extremely challenge due to evanescence nature of these reactive species. An alternative approach to study the involvement of oxidative modification is to detect and quantify the stable molecular fingerprints, like oxidized tyrosine species, in biological matrices. Previously reported analytical methods for quantifying oxidized amino acids have typically been limited by: low sensitivity, specificity and the failure of detection potential oxidation products generated during sample handling. Using GC/MS in combination with stable isotope dilution, a sensitive and self-quality control assay was developed. Using this isotope-dilution GC/MS and multiple allergen challenge models, we demonstrate that EPO is the major pathway for generating protein-bound 3-bromotyrosine in vivo. However, the need for chemical derivatization of samples increases the possibility that artifactual generation of oxidation products which may interfere with the detection of native levels. Requirement of unique derivatization reagent for each oxidized tyrosine results sample analysis both cumbersome and time/labor intensive. Therefore, we develop a liquid chromatography with on-line electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) assay that simultaneously detects and quantifies multiple structurally informative protein oxidation products along with their precursor amino acids. Use of four HPLC systems in multiplex array with column switching permits on-line analyses of only relevant portions of chromatographic profiles and up to four-fold increased throughput efficiency of mass detector usage. Using this high-throughput multiplexed array-based LC-MS/MS method, we found significant correlations among individual oxidized tyrosine in sera from healthy volunteers. These correlations provide chemical
Song, Wei, "Mass Spectrometry-Based High Throughput Approach for Identification of Molecular Modification of Oxidative Process in Respiratory Diseases" (2008). ETD Archive. 277.