Development of a multiplex immunoassay for phosphothreonine protein analysis

Cellular signaling analysis is integral to a thorough understanding of living systems. Cellular analysis methods traditionally have involved the examination of single targets, but new techniques allow for multiplex analysis of proteins. MILLIPLEX® EpiQuant™ technology is an answer to multiplex limitations such as antibody cross‐reactivity and the inability to accurately quantify target protein levels. EpiQuant technology uses computational algorithms to identify unique peptide sequences in proteins. Antibodies generated against these sequences have pre‐defined target specificity. Protein phosphorylation measurements are made at the peptide level, using EpiQuant antibodies, following proteolytic fragmentation of samples. This process has a number of benefits including: a synthetic peptide can be used as a quantitative standard, cross‐reactivity with similar peptide sequences in other proteins can be predicted and avoided and phosphorylated and total protein analysis of a single protein can be conducted in a single well. These attributes result in a superior methodology for cellular signaling multiplex analysis. We have utilized the EpiQuant technology to develop a multiplex assay consisting of 16 phosphothreonine protein targets. These targets include Akt, NFκB and IRS, among others. Here we demonstrate the use of this panel for the analysis of tissue culture lysates.