Elucidation Of Histone Modifications And Nucleosomal Structure Using Novel Mass Spectrometry Approaches

The fundamental repeating unit of chromatin is the nucleosome, composed of 147 base pairs of DNA wrapped around a histone protein octamer containing two copies of H2A, H2B, H3, and H4. Histone proteins are involved in many critical nuclear processes including transcription and maintenance of chromatin structure. Histone function is mediated by a dynamic and extensive array of post-translational modifications (PTMs). Mass spectrometry (MS) has emerged as a leading tool to study these complex histone PTM profiles. Generally, MS experiments utilize data dependent acquisition (DDA) methods on high-resolution MS instruments because they can more readily distinguish PTMs with small mass differences. I demonstrate here that low-resolution instruments are capable of this analysis with data dependent acquisition (DDA) and data independent acquisition (DIA) methods, thereby expanding the repertoire of instruments that can be used. However, DIA methods improve quantification of isobaric peptides compared to DIA and also allows for re-mining of data post-experiment. This dissertation also highlights work I have done to develop MS methods to identify and quantify ADP-ribosylation PTMs, which are critical for DNA damage repair pathways. We identified 30 ADP-ribosylation marks on histones, 20 of which are novel. We quantified 10 of these sites throughout a DNA damage and found that all of these sites increase in abundance over time, indicating that it is unlikely that specific sites are required for repair, but rather that ADP-ribosylation of the nucleosome surface in general is needed. Histone function is also mediated through its structure and dynamic properties. Hydrogen-deuterium exchange (HDX) coupled to MS is a powerful technique to monitor these properties in solution. However, traditional HDX-MS studies on histone proteins were unable to monitor histone N-terminal tail domains, where a majority of PTM sites are located. Here, we demonstrate that by incorporating electron transfer dissociation (ETD) MS/MS methodology with middle-down and top-down MS, we are able to measure deuterium content of tail domains with near site-specific resolution for the first time. We find that all tails undergo decreased structural rigidity upon incorporation into the nucleosome, lending the first detailed experimentally-obtained insight into histone tail structure in solution