Analysis of Histone Antibody Specificity with Peptide Microarrays

Abstract

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

Study of mitotic chromatin supports a model of bookmarking by histone modifications and reveals nucleosome deposition patterns

“…The incorporation of MS data is particularly valuable when assessing histone acetylation levels, since mitosis-specific H3S10 and H3S28 phosphorylations may limit the functionality of H3K9ac and H3K27ac antibodies, thereby affecting the ChIP-seq results (Supplemental Fig. S17; Rothbart et al 2015;Cornett et al 2017). For this reason, we rely on the MS results to evaluate the mitotic levels of these acetylations, rather than on the ChIP-seq data.…”

“…Exactly 2.1 million cells from each sample were snap-frozen in liquid nitrogen and stored at −80°C. Global chromatin profiling (GCP) of cell pellets to quantify the levels of histone modification was performed as described previously (Creech et al 2015). In brief, cells were lysed, nuclei were isolated, and histones were extracted by sulfuric acid and were then precipitated by trichloroacetic acid.…”

“…Prior to MS analysis, a mix of isotopically labeled synthetic peptides was added to each sample. Next, the peptides were separated on a C18 column (15 cm × 75 µm ID, ReproSil-Pur 120 C18-AQ, 1.9 µm) using an EASY-nLC 1000 (Thermo Fisher Scientific) and analyzed using the GCP assay (a parallel reaction monitoring targeted LCMS method) using a Q Exactive-plus mass spectrometer (Thermo Fisher Scientific) as described previously (Creech et al 2015).…”

Study of mitotic chromatin supports a model of bookmarking by histone modifications and reveals nucleosome deposition patterns

“…The incorporation of MS data is particularly valuable when assessing histone acetylation levels, since mitosis-specific H3S10 and H3S28 phosphorylations may limit the functionality of H3K9ac and H3K27ac antibodies ( Figure S17) (Rothbart et al 2015;Cornett et al 2017). For this reason, we rely on the MS results to evaluate the mitotic levels of these acetylations, rather than on the ChIP-seq data.…”

A Degenerate Peptide Library Approach to Reveal Sequence Determinants of Methyllysine-Driven Protein Interactions

“…Peptide microarrays were fabricated using an Aushon 2470 microarrayer and used as described (Cornett et al, 2017) with the following modifications. Protein and antibody hybridization steps were performed in buffer containing 25 mM HEPES pH 7.5, 100 mM NaCl, 0.5% BSA (w/v), and 0.1% NP-40.…”