Abstract
In the era of proteomics, high-throughput screening of posttranslational states of proteins, especially protein modifications such as phosphorylation is considered to be of utmost importance. However, current protein modification detection depends on either the combination of proteolysis and mass spectrometry, or time-consuming immunoassay that requires inevitable washing processes. As a way to rapidly assay protein modification, here we propose the use of Open Sandwich Immunoassay that detects antigen-dependent stabilization of antibody variable region (Fv). As a model, the heavy and light chain variable regions (VH/VL) of anti-phosphotyrosine antibody PY20 were used to evaluate its performance. When measured by phage display method, while wild-type Fv showed modest phosphotyrosine (PY)-dependent stabilization of ∼30%, a mutant with weak VH/VL interaction (HQ39R) showed markedly improved response of >200%. Using this mutant, two fusion proteins in which each variable region fragment was tethered to GFP color variant were made (VH-YFP/VL-CFP), to monitor PY-induced fluorescence resonance energy transfer (FRET) between them. As a result, PY-induced enhancement in FRET was successfully monitored in a homogeneous solution. The result indicates a possibility of rapid screening of tyrosine-phosphorylated proteins in vitro or in situ, and possibly in vivo, opening a way to real-time monitoring of various post-translational events in a cellular system.
