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.

Keywords: HPLC; histones; mass spectrometry; proteoforms; proteomics; top-down.

Figure 1. Five Minute HCl and TCA Precipitations are Sufficient A) HPLC Chromatogram of SUM159 cell line histones with varying HCl precipitation times. Each black line represents a different HCl precipitation time: 5 min, 15min, 30min, 60 min and 120 min. Note that these are not readily distinguishable from each other and overlap almost perfectly. Thus, extensive HCl precipitations, such as overnight, are neither necessary nor beneficial. B) Quantitation of Histones by area under the curve (AUC) with varying HCl precipitation times. The areas under the curve from Figure 1A are integrated for the different time points of HCl precipitation. There is no significant trend for increasing HCl times. C) HPLC Chromatogram of SUM159 histones with varying TCA precipitation times. Each black line represents a different TCA precipitation time: 5 min, 15min, 30min and 60min. There is no difference in retention time or peak heights by increasing TCA precipitation time. D) Quantitation of Histones by AUC with varying TCA precipitation times. The areas under the curve from Figure 1C are integrated for the different time points of TCA precipitation. There is no significant trend for increasing TCA times.

Figure 2 Histone H3 and H4 Standard Curves for Estimating Protein Amount A) Histone H4 Standard Curve. Area of mAU obtained from HPLC analysis of purified histone H4. B) Histone H3.1 Standard Curve. Area of mAU obtained from HPLC analysis of purified histone H3.1.

Figure 3 Quantitative Top Down Proteomic Analysis of Histone H4 Proteoforms A) Histone H4 MS1 extracted ion chromatogram. The peak at 20 min corresponds to oxidized H4, while the dominant peak contains unoxidized H4 proteoforms. B) MS1 scan of +15 charge state histone H4 precursors. C) A single MS2 scan of Histone H4 K20me2.