P81-T Antibody Arrays for Measuring Site-Specific Protein Phosphorylation

We have developed a sandwich immunoassay method for quantitatively measuring site-specific phosphorylation of multiple proteins and at multiple sites within a protein simultaneously, without the need for multiple, phospho-site-specific detection antibodies. This approach utilizes antibodies to capture a unique peptide sequence (EpiTag) proximal to the phosphorylation site of interest. The captured peptides containing the phosphorylation site(s) of interest are then interrogated with a commercially available, pan-anti-phospho antibody to determine whether the site is indeed phosphorylated. Unlike traditional immunoassays, select epitopes are made available to the antibodies by a unique sample-processing procedure. The procedure involves proteolytic fragmentation of proteins in order to segregate multiple phosphorylation sites within a given protein. Since proteolytic cleavage is predictable, quantitation is achieved by interpolating protein concentrations from standard curves generated with synthetic phosphopeptide standards. This approach obviates the need for phosphoproteins, which can be difficult to generate and qualify. The sandwich immunoassays demonstrate titration over a large dynamic range (≥3 logs) and achieve sensitivity at or below picomolar concentrations of standard. In contrast, no sandwich formation is observed with standards that have been dephosphorylated with lambda phosphatase. While the approach is limited to measuring previously discovered phosphorylation sites and does not distinguish between multiple sites that lie within the same proteolytic fragment, this proprietary approach is directly applicable to both bead-based and planar arrays, and has been multiplexed and used to measure >30 phosphotyrosine sites on various targets across multiple signaling pathways. In addition, the approach is currently being evaluated for multiplexed measurements of other phosphoresidues, and we believe this technology can be applied specifically, to study and quantify particular signaling events, as well as on a more global level, to identify and “fingerprint” activated signaling pathways during different disease states or in response to therapeutic strategies.