Cellular effects of gliotoxin: Evaluation of a proteomic, isotope-based method to detect reactive cysteines

Abstract

<p>Cysteinyl residues in proteins are important for many cellular processes and unregulated modification of the cysteine thiol group can have negative effects on cell vitality and viability. In this thesis, the potential for use of the isotope coded affinity tag (ICAT) method for detection of cysteine modification has been investigated. ICAT reagents label free cysteine thiols. The aim of this study was to use HL-60 cells treated with gliotoxin, a fungal metabolite with a reactive disulfide bridge, as a system to evaluate the performance of ICAT for identification of cysteine modification in a whole cell proteome. Gliotoxin has antimicrobial, antitumor, immunosuppressive and cytotoxic properties that have been related to cysteine modification in proteins. Cellular assays including viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, cell cycle analysis, and measurement of reactive oxygen species using dichlorofluorescin diacetate were used to establish conditions for measuring the effects of gliotoxin on HL-60 cells prior to large-scale cellular damage. Cells exposed to gliotoxin and control cells were then labeled with ICAT reagents and analysed by offline reversed phase liquid chromatography followed by matrix-assisted laser desorption/ionization tandem mass spectrometry. The pilot results identified tubulin, glyceraldehyde-3-phosphate dehydrogenase and peptidyl-prolyl cis-trans isomerase as putative targets of gliotoxin. Additionally, this study showed that ICAT can be used to detect modified cysteines from a highly complex sample, but further optimization is needed to unlock the full potential for detection of cysteine modification in complex samples.</p>