Scatter plots comparing two arrays for Rag2, BPTF, and CHD1. Click here to view.(90K, pdf) ACKNOWLEDGEMENTS This work was supported by a National Institutes of Health (NIH) EUREKA award to B.D.S. modifications (left). Supplementary Figure S3. Heat map of all experimental antibody data. Data was normalized to the strongest interaction plotted on a scale from 0 to 1 1 with 1 (yellow) being the most significant. Supplementary Figure S4. Scatter plots comparing two arrays for Rag2, BPTF, and CHD1. NIHMS256681-supplement-2.pdf (90K) GUID:?0D6AEE94-9F2C-4208-BCA6-EC50023B6A08 Summary We report a general method to examine the recognition of post-translational modifications (PTMs) by antibodies and proteins. We use this method to evaluate the binding of modification-specific antibodies and chromatin-associating factors to an array of high-purified, biotinylated peptides (derived from human histone sequences) harboring multiple PTMs printed onto streptavidin-coated glass slides. We find that modification-specific Givinostat antibodies are both more Givinostat promiscuous in their PTM recognition than expected and highly influenced by neighboring PTMs. Binding of chromatin-associating factors is also influenced by combinatorial PTMs, giving further support for the Histone Code hypothesis. Thus we report the first thorough characterization of PTM influence on antibody recognition and describe a tool for the rapid and inexpensive assessment of chromatin-associating factor binding specificity. Results and Discussion Post-translational modifications (PTMs) of proteins such as phosphorylation, methylation, acetylation, and ubiquitination regulate many processes such as protein degradation, protein trafficking, and mediation of protein-protein interactions[1]. Perhaps the best-studied PTMs are those found associated with histone proteins. More than one hundred histone PTMs have been described and they largely function by recruiting protein factors to chromatin, which in turn, drive processes such as transcription, replication, and DNA repair[2]. Likewise, dozens of chromatin-associating factors have been identified that bind to particular histone PTMs and hundreds of modification-specific histone antibodies have been developed to understand the function of these modifications[3]. The enormous number of potential combinations of histone PTMs represents a major obstacle toward our understanding of how PTMs regulate chromatin-templated processes, as well as our ability to develop high-quality diagnostic tools for chromatin and epigenetic studies. The same obstacle applies to other proteins regulated by combinatorial PTMs C for example, p53, RNA polymerase, or nuclear receptors[4-6]. To that end, we developed a peptide array-based S1PR2 platform to begin to address how both proteins and antibodies recognize combinations of PTMs. We focused primarily on the recognition of PTMs associated with the N-terminal tail of histone H3, but this approach is useful for the study of other histone modifications and combinatorial PTMs found on other proteins. We generated a library of 110 synthetic histone peptides bearing either single or combinatorial PTMs and a biotin moiety for immobilization (Figure 1 and Table S2). Prior to printing, all peptides were subjected to rigorous quality control to verify their accuracy (see http://www.med.unc.edu/~bstrahl/Arrays/index.htm for complete details). This is significant, as extensive peptide purification and mass spectrometric analysis is not possible with other recently described array technologies used to study combinatorial histone PTMs[7]. Another significant advancement in our method was the introduction of a biotinylated fluorescent tracer molecule, which served as a positive control for the quality of our printing in all experiments. Givinostat Lastly, peptides were printed as a series of 6 spots, two times per slide by two different pins, yielding 24 independent measurements of every binding interaction per slide. These measures were adopted to minimize binding artifacts due to pin variation or inconsistencies on slide surface. Thus, these arrays and the technical approaches described herein are the first to offer a large number of extensively characterized histone peptide substrates suitable for the assessment of protein or antibody binding. Open in a separate window Figure.