Affi-BAMS™: A Robust Targeted Proteomics Microarray Platform to Measure Histone Post-Translational Modifications

Abstract

For targeted protein panels, the ability to specifically assay post-translational modifications (PTMs) in a quantitative, sensitive, and straightforward manner would substantially advance biological and pharmacological studies. The present study highlights the effectiveness of the Affi-BAMS™ epitope-directed affinity bead capture/MALDI MS platform for quantitatively defining complex PTM marks of H3 and H4 histones. Using H3 and H4 histone peptides and isotopically labelled derivatives, this affinity bead and MALDI MS platform achieves a range of >3 orders of magnitude with a technical precision CV of <5%. Using nuclear cellular lysates, Affi-BAMS PTM-peptide capture resolves heterogeneous histone N-terminal PTMs with as little as 100 µg of starting material. In an HDAC inhibitor and MCF7 cell line model, the ability to monitor dynamic histone H3 acetylation and methylation events is further demonstrated (including SILAC quantification). Affi-BAMS (and its capacity for the multiplexing of samples and target PTM-proteins) thus provides a uniquely efficient and effective approach for analyzing dynamic epigenetic histone marks, which is critical for the regulation of chromatin structure and gene expression.