Jones R. Phosphorylation of proteins is an integral part of the signal transduction of eukaryotic cells as it modulates the activity of complex protein networks. Although Western blot- and immunoprecipitation-based MS approaches (1, 2) can lead to detailed insights into these processes, most of the integrated approaches only allow a static view of protein phosphorylation because they are not suitable for the screening of hundreds of samples. Either planar or bead array-based sandwich immunoassays can be used to analyze the quantity and activation state of signaling molecules in multiplex, enabling the systematic profiling of protein abundance and post-translational modifications (3C6) in hundreds SYN-115 (Tozadenant) of samples. However, multiplex immunoassays are only suitable for the simultaneous analysis of a limited number of proteins. The detection of comprehensive phosphorylation patterns is difficult as this involves assay systems that are incompatible with multiplexing. In principle, Rabbit Polyclonal to OR2AG1/2 two sandwich immunoassay setups are possible for probing the phosphorylation state of a protein. The first setup SYN-115 (Tozadenant) applies a capture antibody specific for a non-modified part of the protein and uses a phosphorylation state-specific detection antibody. When applied to an array-based format, however, this setup does not allow for the simultaneous measurement of the abundance and the degree of phosphorylation (3, 4). A mixture of detection antibodies, one specific for the phosphorylation site and one specific for the non-modified site of the protein, would bind simultaneously to the two different epitopes, and assay signals could not be further deconvoluted by the spatial or color code of the array. The second sandwich immunoassay setup for the analysis of protein phosphorylation applies a phosphorylation state-specific capture antibody and a protein-specific detection antibody. In such a setup, an anti-phosphotyrosine antibody (mAb 4G10) cannot be applied as a capture antibody because a huge variety of tyrosine phosphorylated proteins would be captured, and specific signals could rarely be deconvoluted. Using capture antibodies that bind to phosphorylated epitopes in the context of their flanking amino acids is not a problem until a multiplex readout is desired. If one antibody specific for the phosphosite and one antibody specific for the abundance of a protein SYN-115 (Tozadenant) are used together in a multiplex assay panel they might compete for their analyte. The situation becomes even more complex if the protein of interest contains various phosphorylation sites such as the epidermal growth factor receptor. Several capture antibodies target different epitopes of the same protein and therefore compete for the overall amount of targeted protein in the sample, thus making a valid simultaneous measurement problematic. Although different ways of tackling the problem of assay multiplexing are in use, we demonstrated the feasibility to sequentially perform such incompatible assays from the same sample using a magnetic particle handler that moves particles through the samples and reagents (Fig. 1). Using a model assay, we confirmed that suspension bead array-based immunoassays work under ambient analyte conditions. As described by Roger Ekins (7), decreasing of the amount of capture antibody in a sandwich immunoassay setup from a macrospot (a microtiter plate assay) to a microspot generates a scenario where only a tiny fraction of the present target analytes is captured on the microspot. Therefore, the overall concentration of the analyte molecules in the sample does not change significantly even in the case of low target concentrations and high affinity binding reactions. Furthermore, as the initial concentration of the analyte is not significantly changed when performing a miniaturized sandwich immunoassay, multiple post-translational modifications within the same protein can be measured either in sequence or in parallel in the same multiplex panel. Open in a separate window Fig. 1. Sequential multiplex analyte capturing. Magnetic suspension bead array assays can be performed.