Quantitative Analysis of Chromatin Proteomes in Disease

Summary

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

Abstract

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,¹ the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.² Transcriptional regulation during development and disease have been well studied in this organ,^3-5 but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.⁶ In the developed world, heart disease is the number one cause of mortality for both men and women.⁷ Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options.

Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,^8-13 until recently¹⁴ there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.¹⁵ In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.¹⁶ Additionally, cardiomyocytes are 40% mitochondria by volume¹⁷ which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo experimentation in various animal models and organ systems where metabolic labeling is not feasible.

Protocol

The experimental workflow contains seven major steps (Figure 1). For any work involving samples that will be run on the mass spectrometer, the experimenter should wear a lab coat, gloves and hair net and take care to avoid contamination from dust and personal shedding of keratin.

1. Heart Homogenization and Nuclear Isolation

Mouse hearts are homogenized and an intact nuclei pellet is isolated (Figure 2).

1. Sacrifice adult mouse, excise the heart, rinse in ice-cold PBS, and homogenize on ice in glass dounce (we prefer the Wheaton Tissue Grinder from Fisher, #08-414-13A, ....

Results

Figure 4 highlights the utility of this form of relative quantification. Shown in the left panel are the individual monoisotopic peptide peaks (overlaid from different mice), which have been designated as belonging to the protein HMGB1 (identified via database search). Each peak, essentially an extracted ion chromatograph for the given peptide, comes from a different mouse. The groups represent three different physiological states: basal, cardiac hypertrophy, and heart failure, with three biological repl.......

Discussion

Two main methods for nuclear isolation have been reviewed previously:²⁷ one is the Behrens technique of homogenizing lyophilized tissue in a non-aqueous solvent and the second, a modification of which we use here, of homogenizing tissue in an aqueous sucrose/salt solution followed by differential or density-gradient centrifugation.

Subfractionation of the nuclei by acid extraction on tissue samples is an important tool for studying chromatin which has been used since 1960,²⁸

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbeco Modified Eagle Medium	Invitrogen	11965
Protease pellet	Roche	04 693 159 001
100 μm strainer	BD Falcon	352360
Ultracut ultramicrotome	Reichert
100CX Transmission Electron Microscope	JEOL USA, Inc.
Oriole	BioRad	161-0496
Histone H2A antibody	Santa Cruz	sc-8648
Nucleoporin p62 antibody	BD Biosciences	610498
Adenine nucleotide transporter antibody	Santa Cruz	sc-9299
BiP antibody	Santa Cruz	sc-1050
Tubulin antibody	Sigma	T1568
Histone H3 antibody	Abcam	ab1791
Fibrillarin antibody	Cell Signaling	C12C3
SNRP70 antibody	Abcam	ab51266
E2F-1 antibody	Thermo Fisher	MS-879
Retinoblastoma antibody	BD Biosciences	554136
Hypoxia inducible factor-1 antibody	Novus Biologicals	NB100-469
BCA protein assay	Thermo Scientific	23227
Reverse phase column	New Objective	PFC7515-B14-10
BioWorks Browser	Thermo Scientific