Multiplexed Proteomics Technology for the Fluorescence Detection of Phosphorylation and Protein Expression Levels Using Pro-Q® Diamond and SYPRO® Ruby Dyes

Abstract

The reversible phosphorylation of serine, threonine, and tyrosine residues is arguably one of the most important covalent posttranslational modifications regulating the functional status of proteins in eukaryotic organisms. Tools and techniques for determining the phosphorylation status of proteins and peptides thus play a prominent role in the investigation of diverse biological phenomena, including signal transduction, cell division, cell motility, apoptosis, metabolism, differentiation, gene regulation, and carcinogenesis. Typically, cells or isolated proteins are labeled with 32 P or 33 P prior to gel electrophoresis using protein kinases (1). The phosphoproteins are then usually detected by autoradiography, using film or storage phosphor imaging screens. In vitro radiolabeling provides a measure only of the phosphate groups attached during the actual labeling. No information is provided with respect to the physiological phosphorylation status of the proteins (2). Radiolabeling phosphoproteins also presents safety issues associated with handling the material and contamination of instrumentation (3).