[Abstract]  [Full Text]  [PDF]

H. Fai Poon¹, Laila Abdullah¹, Jon Reed¹, Sarah M Doore¹, Cyndi Laird¹, Venkat Mathura¹, Michael Mullan¹ and Fiona Crawford¹

¹ Roskamp Institute. Sarasota, FL 34243. USA.

* To whom correspondence should be addressed: H. Fai Poon, Roskamp Institute. 2040 Whitfield Ave., Sarasota, FL 34243. USA. Phone: +941 752 2949. Fax: +941 752 2948. Email: faipoon@rfdn.org

Biol. Proced. Online 2007;9:65-72. doi:10.1251/bpo134
Submitted: August 31, 2007; Accepted: December 09, 2007; Published: December 19, 2007.

Indexing terms: electrophoresis, gel, two-dimensional; oxidation-reduction; protein processing, post-translational.

Abbreviations: FHA, fluorescent hydroxylamine; IEF, isoelectric focusing; MALDI, Matrix-assisted laser desorption/ionization.

Recent advances in redox proteomics have provided significant insight into the role of oxidative modifications in cellular signalling and metabolism. At present, these techniques rely heavily on Western blots to visualize the oxidative modification and corresponding two dimensional (2D) gels for detection of total protein levels, resulting in the duplication of efforts. A major limitation associated with this methodology includes problematic matching up of gels and blots due to the differences in processing and/or image acquisition. In this study, we present a new method which allows detection of protein oxidation and total protein on the same gel to improve matching in image analysis. Furthermore, the digested protein spots are compatible with standard MALDI mass spectrometry protein identification. The methodology highlighted here may be useful in facilitating the development of biomarkers, assessing potential therapeutic targets and elucidating new mechanisms of redox signalling in redox-related conditions.