Bead-based peptide arrays for profiling the specificity of modification state-specific antibodies

Abstract

Modification state-specific antibodies are powerful tools for investigating posttranslational modifications in proteins. The majority of these antibodies have been generated against peptide-antigen conjugates. They are useful in a plethora of methods, such as Western blotting, immunohistochemistry, sandwich immunoassay, immunoprecipitation, and immunoprecipitation coupled with mass spectrometry. Phosphorylation, acetylation, methylation, sulfation, nitrosylation, ubiquitination, and sumoylation are some of the modifications that can be studied using such antibodies. However, investigating the on- and off-target binding of antibodies is crucial to the interpretation of experimental data. Peptide arrays are excellent tools for such in-depth studies of off-target and on-target binding of antibodies. Dozens or even hundreds of modified peptides can be integrated into a single experimental setup to analyze the antibody’s binding behavior. Here, we propose three different protocols for peptide bead array generation and describe their suitability for such types of assay.