Proteomics in chromatin biology and epigenetics: Elucidation of post-translational modifications of histone proteins by mass spectrometry

Sidoli, Simone; Cheng, Lei; Jensen, Ole N.

doi:10.1016/j.jprot.2011.12.029

Cited by 119 publications

(101 citation statements)

References 156 publications

(159 reference statements)

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“…Mass spectrometry (MS)-based strategies have continuously evolved toward higher throughput and flexibility, allowing not only identification and quantification of single histone PTMs, but also their combinatorial patterns and even characterization of the intact proteins (reviewed in (6,7)). For histone analysis, a widely adopted workflow for nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) includes derivatization of lysine residue side chains with propionic anhydride, proteolytic digestion with trypsin, and subsequent derivatization of peptide N termini (8,9).…”

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confidence: 99%

Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH) Analysis for Characterization and Quantification of Histone Post-translational Modifications

Sidoli

Lin

Xiong³

et al. 2015

Molecular & Cellular Proteomics

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Histone post-translational modifications (PTMs) have a fundamental function in chromatin biology, as they model chromatin structure and recruit enzymes involved in gene regulation, DNA repair, and chromosome condensation. High throughput characterization of histone PTMs is mostly performed by using nano-liquid chromatography coupled to mass spectrometry. However, limitations in speed and stochastic sampling of data dependent acquisition methods in MS lead to incomplete discrimination of isobaric peptides and loss of low abundant species. In this work, we analyzed histone PTMs with a data-independent acquisition method, namely SWATH™ analysis. This approach allows for MS/MS-based quantification of all analytes without upfront assay development and no issues of biased and incomplete sampling. We purified histone proteins from human embryonic stem cells and mouse trophoblast stem cells before and after differentiation, and prepared them for MS analysis using the propionic anhydride protocol. Results on histone H3 peptides verified that sequential window acquisition of all theoretical mass spectra could accurately quantify peptides (<9% average coefficient of variation, CV) over four orders of magnitude, and we could discriminate isobaric and co-eluting peptides (e.g. H3K18ac and H3K23ac) using MS/MS-based quantification. This method provided high sensitivity and precision, supported by the fact that we could find significant differences for remarkably low abundance PTMs such as H3K9me2S10ph (relative abundance <0.02%). We performed relative quantification for few sample peptides using different fragment ions and observed high consistency (CV <15%) between the fragments. This indicated that different fragment ions can be used independently to achieve the same peptide relative quantification. Taken together, sequential window acquisition of all theoretical mass spectra proved to be an easy-to-use MS acquisition method to perform high quality MS/MS-based quantification of histone-modified peptides. Molecular & Cellular

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confidence: 99%

Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH) Analysis for Characterization and Quantification of Histone Post-translational Modifications

Sidoli

Lin

Xiong³

et al. 2015

Molecular & Cellular Proteomics

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“…The first issue can be solved by combining this technique with a middle-down or top-down approach (reviewed in 16 ). This type of analysis, even if technically more challenging, is ideal for studying co-existence frequencies of modifications.…”

Section: Discussionmentioning

confidence: 99%

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Sidoli

Bhanu

Karch

et al. 2016

JoVE

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174

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Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 -20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

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“…Although advantageous for their sensitivity, antibody-based assays are limited in their capability to distinguish similar modifications and in dissecting the combinatorial aspect of the histone code 36 . On the other hand, while MS provides a comprehensive and unbiased analysis of hPTMs, singly and in combinations 9 , it has so far been applied to the analysis of bulk chromatin, with consequent lack of information on locus-specific PTMs patters. We employ a modified version of ChIP to isolate functionally distinct chromatin domains and mass spectrometry to characterize the histone PTM patterns and the non-histonic proteins specifically co-enriched.…”

Section: Discussionmentioning

confidence: 99%

“…MS detects a modification as a specific Δmass between the experimental and theoretical mass of a peptide. At the level of individual histones, MS provides an unbiased and comprehensive method to map PTMs, allowing the detection of new modifications and revealing interplays among them [9][10][11][12][13][14] .…”

Section: Introductionmentioning

confidence: 99%

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Soldi

Bonaldi

2014

JoVE

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Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locusspecific structural and functional configurations. Video LinkThe video component of this article can be found at

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Proteomics in chromatin biology and epigenetics: Elucidation of post-translational modifications of histone proteins by mass spectrometry

Cited by 119 publications

References 156 publications

Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH) Analysis for Characterization and Quantification of Histone Post-translational Modifications

Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH) Analysis for Characterization and Quantification of Histone Post-translational Modifications

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

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