Expeditious Extraction of Histones from Limited Cells or Tissue Samples and Quantitative Top‐Down Proteomic Analysis

Abstract

Histones are the primary protein component of chromatin and are involved in virtually all DNA‐templated processes. Histones are abundantly post‐translationally modified by a variety of chromatin‐modifying machinery. These post‐translational modifications (PTMs) are recognized by a range of “reader” proteins, which recruit additional proteins to specific locations on chromatin and impart precise and powerful effects on gene regulation. Each PTM typically exerts a positive or negative effect on transcription, and recent studies have shown that histone PTMs function in a combinatorial histone code: that is, histone PTMs function in combination to exert precise DNA‐templated regulation. Thus, there is a need to identify and understand proteoforms, or unambiguously defined single protein molecules with all combinations of modifications. Top‐down proteomics is currently the only viable approach for identifying and quantitating histone proteoforms, and mass spectrometry instruments have become sufficiently powerful to perform these quantitative analyses in a robust and high‐throughput fashion. These recent innovations have enabled new experimental directions in chromatin research but have also introduced temporal and other constraints. This has led us to develop the protocols described here, which increase throughput, reduce sample requirements, and maintain robust quantitation. Although originally designed for high‐throughput quantitative top‐down proteomics, the protocols described here are useful for a wide range of chromatin biology applications. Starting with small amounts of cells or tissue, we describe two basic protocols for exceptionally rapid and efficient nuclei isolation, acid extraction of histones, and high‐performance liquid chromatography fractionation of histones into histone families. We additionally describe the quantitative top‐down proteomic analysis of histone H4 proteoforms. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Nuclei isolation and acid extraction of histones from mammalian cells in culture/tissues Basic Protocol 2: HPLC fractionation of histones and histone H4 HPLC‐MS/MS Support Protocol: Preparation of intact H3 histone tails by Glu‐C digestion

An acetylation-mediated chromatin switch governs H3K4 methylation read-write capability

“…The above data suggested a molecular model for how the H3 N-terminal tail, via cis acetylation, becomes available for H3K4 reader binding or enzymatic modification in vitro. To determine if such acetylation could function as an accessibility switch in vivo , we used middle-down mass spectrometry on acid-extracted histones from asynchronous MCF-7 breast cancer cells to measure the relationship between H3K4 methylation and tail acetylation on the same H3 proteoforms (Holt et al, 2021; Smith and Kelleher, 2013). As expected (Garcia et al, 2007; Peach et al, 2012; Young et al, 2009), the absolute amounts of H3K4me3 and higher Kac states (3ac, 4ac, and 5ac) across adjacent lysine residues were extremely low ( Figure S3A ).…”

“…Cells were washed with cold PBS (11.9 mM phosphates, 137 mM NaCl, 2.7 mM KCl) to remove residual sodium butyrate, harvested by scraping, and flash frozen in liquid nitrogen. Histones were acid extracted after nuclei isolation as described (Holt et al, 2021). Isolated histones were resuspended in 85 μL 5% acetonitrile, 0.2% trifluoroacetic acid (TFA) and resolved by offline high-performance liquid chromatography (HPLC) as described (Holt et al, 2021).…”

“…Histones were acid extracted after nuclei isolation as described (Holt et al, 2021). Isolated histones were resuspended in 85 μL 5% acetonitrile, 0.2% trifluoroacetic acid (TFA) and resolved by offline high-performance liquid chromatography (HPLC) as described (Holt et al, 2021). Reverse Phase HPLC fractionation was performed with a Thermo U3000 HPLC system ( ThermoFisher ) with a 150 × 2.1–mm Vydac 218TP 3 μm C18 column ( HiChrom # 218TP3215), at a flowrate of 0.2 mL/min using a linear gradient from 25% B to 60% B in 60 min.…”

“…The EC80 rel was selected as the optimal probing concentration for discovery screens because of the robust signal-to-background and to provide the best opportunity to bind targets without saturating the primary target signal. To compute EC80 rel values, we used the formula ECF rel = (F/ (100 -F) as described (Holt et al, 2021). Reverse Phase HPLC fractionation was performed with a Thermo U3000 HPLC system (ThermoFisher) with a 150 × 2.1-mm Vydac 218TP 3 µm C18 column (HiChrom # 218TP3215), at a flowrate of 0.2 mL/min using a linear gradient from 25% B to 60%…”

Novel Strategies to Address the Challenges in Top-Down Proteomics

“…Nonetheless, it should be noted that certain protein classes are currently more amenable to acid/base extractions, such as acid-soluble histones. 76,77 MS-based proteomics experiments are commonly performed under denaturing conditions to obtain the primary structural analysis of proteins. 78 In contrast, native MS performed under non-denaturing conditions seeks to preserve whole protein structures and their complexes, which enables the study of protein−protein interactions and ligand/drug binding.…”