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Streamlined discovery of cross-linked chromatin complexes and associated histone modifications by mass spectrometry

Significance Scientists have developed many affinity pulldown methods to characterize protein networks. Three persistent challenges with current approaches are capturing labile interactions, isolating insoluble DNA-bound complexes, and preserving modifications potentially important for binding specificity. This report presents a cross-linked affinity approach to meet these challenges and effectively capture interactions that otherwise may be intractable with native affinity techniques. We isolate several Drosophila and human protein complexes to demonstrate the technique’s scalability across a range of initial cell amounts and sources, and we develop the ability to identify the breadth of associated histone and nonhistone modifications in parallel. Our results indicate that cross-linked affinity pulldowns should be broadly useful for comprehensive analyses of chromatin networks. Posttranslational modifications (PTMs) are key contributors to chromatin function. The ability to comprehensively link specific histone PTMs with specific chromatin factors would be an important advance in understanding the functions and genomic targeting mechanisms of those factors. We recently introduced a cross-linked affinity technique, BioTAP-XL, to identify chromatin-bound protein interactions that can be difficult to capture with native affinity techniques. However, BioTAP-XL was not strictly compatible with similarly comprehensive analyses of associated histone PTMs. Here we advance BioTAP-XL by demonstrating the ability to quantify histone PTMs linked to specific chromatin factors in parallel with the ability to identify nonhistone binding partners. Furthermore we demonstrate that the initially published quantity of starting material can be scaled down orders of magnitude without loss in proteomic sensitivity. We also integrate hydrophilic interaction chromatography to mitigate detergent carryover and improve liquid chromatography-mass spectrometric performance. In summary, we greatly extend the practicality of BioTAP-XL to enable comprehensive identification of protein complexes and their local chromatin environment.