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

Abstract

Protein glycosylation is increasingly being recognized as one of the most prominent posttranslational modifications associated with malignant transformation and tumorogenesis as well as cell differentiation. The multiplexed proteomics platform is a new technology that permits quantitative, multicolor fluorescence detection of proteins in gels and on Western blots. This methodology allows the sequential determination of both protein expression level changes as well as altered glycosylation patterns within a single experiment. The linear responses of the fluorescent dyes allow rigorous quantitation of changes in protein expression using advanced image analysis software over a broad 3-log linear dynamic range. Global analysis of glycosylation changes can be expanded by dichromatic lectin-based profiling methods for rapidly categorizing glycan branching structure as well as deglycosylation studies employing exo- and endoglycosidases (1,2. The basic principles and methodologies for the multiplexed proteomics technology are explained in this chapter.