| Oxidative stress is a kind of metabolomic disorder that can cause reduced thiols to form disulfide bonds. Therefore, relative quantitation of reduced thiol metabolites can help to investigate the oxidized stress. Maleimide tagging of thiol metabolites has the advantage of a short reaction time and identification of thiols based on mass and retention time. In this thesis, it is shown that by using tags of similar hydrophobicities but different masses, thiols from complex biological samples can be analyzed and separated with HILIC-MS. Combined butyl-type tagged thiols in the control sample and hexyl-type tagged thiols in the experimental sample in a 80% acetonitrile solution under alkaline conditions can detect the different signal intensity by overlaps, relatively quantitate the thiols concentration, and investigate oxidation activity in the sample. After obtaining the calibration curve from thiol standards, both cell culture solution and heart tissue were successfully tested by this ratiometric approach. The purpose of this work is to develop a tagging method for identification, relative quantitation, and separation of thiol metabolites without the use of isotopes. This work will benefit understanding of relative therapeutic methods and the pathology resulting from oxidative stress. |
