Oral Presentation 23rd Annual Lorne Proteomics Symposium 2018

Exploring thiol proteomes with maleimide-based probes and quantitative proteomics (#3)

Marisa Duong 1 2
  1. Proteomics International, Nedlands, WA, Australia
  2. School of Molecular Sciences, The University of Western Australia, Crawley, Western Australia, Australia

The X-linked degenerative muscular disease Duchenne Muscular Dystrophy (DMD) affects about 1 in 3500 boys worldwide. Although pathological mechanisms remain unclear, reactive oxygen species appear to be pivotal in exacerbating disease progression. In DMD, we hypothesise that oxidation of the thiol functional groups (-SH) of cysteine residues is affecting the function of proteins to cause cellular and tissue pathology. Through preliminary electrophoretic work with fluorescent maleimide thiol probes, several proteins were found to be more thiol oxidised in muscles of the mdx mouse model of DMD compared with normal mice. To identify proteins undergoing thiol oxidation, we synthesised customised maleimide-based isotopic (2H) mass tags. The identified protein list included titin, a structural protein directly involved in muscle contractile machinery. Titin was oxidised at specific thiol groups that had functional relevance to the weakening of muscle contraction in DMD. My objective is to improve sample preparation techniques and methods of mass spectrometry analysis to identify less abundant muscle proteins undergoing thiol oxidation. We have now synthesised 13C maleimide probes to achieve chromatographic coelution for liquid chromatography mass spectrometry and hence enhance the ability to quantify the amount of thiol oxidation. These probes can also be used with Matrix Assisted Laser Desorption/Ionization (MALDI), which provides a high throughput platform to optimise proteomic workflow for sample preparation. Using MALDI, I have established that standard curves generated with the 13C maleimide probes are not affected by common desalting methods (C18 Ziptip and StrataX). These proteomic techniques can be used in combination to maximise the ability to identify and quantify the oxidation of less abundant proteins.