Simultaneous Proteoform Analysis of Histones H3 and H4 with a Simplified Middle-Down Proteomics Method

Abstract

Dynamic post-translational modifications of histones regulate transcriptional gene expression in eukaryotes. Unique combinations of modifications, almost exclusively displayed at the flexible N-terminal tails on histones, create distributions of proteoforms that need to be characterized in order to understand the complexity of gene regulation and how aberrant modification patterns influence disease. Although mass spectrometry is a preferred method for the analysis of histone modifications, information is lost when using conventional trypsin-based histone methods. Newer "middle-down" protocols may retain a greater fraction of the full proteoform distribution. We describe a strategy for the simultaneous characterization of histones H3 and H4 with near-complete retention of proteoform distributions, using a conventional proteomics liquid chromatography-tandem mass spectrometry (LC-MS/MS) configuration. The selective prolyl endoprotease neprosin generates convenient peptide lengths for retention and dispersion of modified H3 and H4 peptides on reversed-phase chromatography, offering an alternative to the hydrophilic interaction liquid chromatography typically used in middle-down methods. No chemical derivatizations are required, presenting a significant advantage over the trypsin-based protocol. Over 200 proteoforms can be readily profiled in a single analysis of histones from HeLa S3 cells. An in-gel digestion protocol provides additional options for effective histone analysis.

The CHD6 chromatin remodeler is an oxidative DNA damage response factor

“…Samples were desalted using C18 ZipTips prior to nano liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. All histone digests were analyzed on an Orbitrap Velos mass spectrometer coupled to an EASY-nLC 1000 system (Thermo Fisher Scientific, Bremen, Germany) equipped with a Nanospray Flex ion source as it has been described elsewhere 64 . The flow rate was set to 300 nL/min.…”

Accelerating the Field of Epigenetic Histone Modification Through Mass Spectrometry–Based Approaches

“…used an alternative protease, Neprosin, to generate H3 peptide 1-38 AA as the most abundant H3 digestion product which allows better retention and dispersion than H3 N-tail 1-50 AA on reversed-phase chromatography (23). Janssen et al used porous graphitic carbon (PGC) as a novel stationary phase and achieved simultaneous bottom-up and middle-down histone analysis using the same reversed-phase buffer setup (31).…”

Middle-down approach: a choice to sequence and characterize proteins/proteomes by mass spectrometry

“…76,184 Very recently, Schräder et al have reported MD strategy by using neprosin, a novel propylendoprotease for getting the middle-range sized peptides to characterize the H3 and H4 histones of HeLa S3 cells. 267 It needs to be noted that BU approach may not be suitable to study histones, especially their PTMs. 77,268 This is because N-terminal tails of histones are rich in lysine and arginine residues and therefore, very short length peptides would be produced upon carrying out trypsin digestion.…”