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

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.

Complementary PTM Profiling of Drug Response in Human Gastric Carcinoma by Immunoaffinity and IMAC Methods with Total Proteome Analysis.

Gaining insight into normal cellular signaling and disease biology is a critical goal of proteomic analyses. The ability to perform these studies successfully to extract the maximum value and discovery of biologically relevant candidate biomarkers is therefore of primary importance. Many successful studies in the past have focused on total proteome analysis (changes at the protein level) combined with phosphorylation analysis by metal affinity enrichment (changes at the PTM level). Here, we use the gastric carcinoma cell line MKN-45 treated with the c-Met inhibitor SU11274 and PKC inhibitor staurosporine to investigate the most efficient and most comprehensive strategies for both total protein and PTM analysis. Under the conditions used, total protein analysis yielded few changes in response to either compound, while analysis of phosphorylation identified thousands of sites that changed differentially between the two treatments. Both metal affinity and antibody-based enrichments were used to assess phosphopeptide changes, and the data generated by the two methods was largely complementary (non-overlapping). Label-free quantitation of peptide peak abundances was used to accurately determine fold-changes between control and treated samples. Protein interaction network analysis allowed the data to be placed in a biologically relevant context, and follow-up validation of selected findings confirmed the accuracy of the proteomic data. Together, this study provides a framework for start-to-finish proteomic analysis of any experimental system under investigation to maximize the value of the proteomic study and yield the best chance for uncovering actionable target candidates.