Detection of Post-translational Modifications by Fluorescent Staining of Two-Dimensional Gels

Abstract

Post-translational modifications (PTMs) are key to the regulation of functional activities of proteins. Quantitative and qualitative information about PTM stages of proteins is crucial in the discovery of biomarkers of disease. Recent commercial availability of fluorescent dyes specifically staining PTMs of proteins such as phosphorylation and glycosylation enables the specific detection of protein regulations taking place with respect to these modifications. Activity and molecular and signalling interactions of many proteins are determined by their extent of phosphorylation. In our search for biomarkers of neurodegenerative diseases such as Multiple Sclerosis (MS), using its animal model, Experimental autoimmune encephalomyelitis (EAE), we have applied the phopshorylation specific fluorescent dye, ProQ Diamond, to study changes taking place in the phosphoproteome. Subsequent Colloidal Coomassie staining of the same gels detects the changes at the whole proteome level. We have detected many changes taking place in the CNS tissue of the EAE animals at the whole proteome as well as at the phosphoproteome level that has given valuable insights into the patho-physiological mechanism of EAE and possibly also MS.