Detection of Posttranslational Modifications by Fluorescent Staining of Two-Dimensional Gels

Posttranslational modifications (PTMs) are key to the regulation of functional activities of proteins. Quantitative and qualitative information about PTM stages of proteins is crucial for the discovery of disease biomarkers. Fluorescent dyes specifically staining protein PTMs such as phosphorylation and glycosylation enable the specific detection of protein regulations taking place with respect to these modifications. Activity and molecular 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 an animal model, experimental autoimmune encephalomyelitis (EAE), we have applied the phosphorylation-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 resulting in valuable insights into the pathophysiological mechanism of EAE and MS.