Identification Of Binding Proteins By Self-Assembled Photoaffinity Peptide Probes And Quantitative Mass Spectrometry

Histone post-translational modifications(HPTMs)is an important component of epigenetics.It can regulate the transcription of genes and determine the fate of cells.Dysregulation of PTMs is closely related to the occurrence and development of many major human diseases.It is widely believed that one of the important ways for histone modification to function is to recruit readers to regulate many biological activities,so the identification of HPTM readers is essential to understand the mechanism of epigenetic regulation.However,since HPTMs are highly dynamic,the abundance of readers is usually low,and the interactions between PTMs and its readers are quite weak and transient,it is still a big challenge to find new PTM readers.This hinders people's deep understanding of the modifications,especially for the new modifications.Here,we develop a kind of self-assembled multivalent photoaffinity peptide probes.We fix peptide and photo-cross-linker to the surface of AuNPs by self-assembled(SAM)technique.The peptides serve as a bait and the photo-cross-linker helps to convert the weak interactions between PTMs and readers to strong covalent interactions.Polyethylene glycol(PEG),the support of the photo-cross-linker,can be adjusted to a proper length and amount to get a better cross-linking efficiency.Combination of probe affinity enrichment with quantitative MS,we use modified and unmodified peptide probes to pull-down from nuclear extracts,and a quantitative filter to distinguish specific PTM readers from the vast amount of background binders that are typically present.We first applied this approach to a well-studied HPTM,H3K4me3,and we successfully identified some known H3K4me3 direct and indirect binding partners,which verified the feasibility of this approach.Histone lysine ?-hydroxybutyrylation,2-hydroxyisobutyrylation,and crotonylation are all acylation modifications identified in recent years.Among these modifications,?-hydroxybutyrylation is the latest to be found.It cannot alter the charge state of histone,so it most likely to exert biological functions by recruiting readers.Therefore,we applied this approach to H3K9 bhb,and identified the potential binder ENL of H3K9 bhb.Then we verified the accuracy of MS quantitative results by PRM and western blot and we docked the binding of ENL with the peptide K9 bhb to elucidate the molecular basis for H3K9 bhb readout by ENL.Similar workflows are also applied to H3K9 hib and H3K9 cr,and we identified known readers and unreported potential readers of these PTMs.We also used bioinformatics tools to analysis molecular functions,cellular components,and biological processes of these binders.Besides,we compared the differences among these binders of the three pairs of probes due to the structural similarities of the three modifications.The experimental results show that our self-assembled multivalent photoaffinity peptide probe affinity enrichment combined with proteomic quantification has important value in studying the interaction between histone modifications and their readers.