Liquid chromatography mass spectrometry profiling of histones

Su, Xiaodan; Jacob, Naduparambil K.; Amunugama, Ravindra; Lucas, David M.; Knapp, Amy R.; Ren, Chen; Davis, Melanie E.; Marcucci, Guido; Parthun, Mark R.; Byrd, John C.; Fishel, Richard; Freitas, Michael A.

doi:10.1016/j.jchromb.2006.12.037

Cited by 29 publications

(46 citation statements)

References 61 publications

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“…Su and colleagues recently demonstrated the use of LC/MS using a custom-manufactured C18 column for an improved separation of histones. The column was coupled to an ESI time-of-flight (TOF) mass spectrometer for the routine and fast screening analysis of histones, which can identify the changes of modified isoforms and/or histone variants between different cell lines or after drug treatment [95]. Consequently, a complete characterization of both core and linker histones was achieved and the results suggest that histones H2A and H2B are present as both modified forms and sequence variants.…”

Section: Reverse-phase High-performance Liquid Chromatography Mass Smentioning

confidence: 99%

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

Ren

Freitas

2007

Expert Review of Proteomics

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Due to the intimate interactions between histones and DNA, the characterization of histones has become the focus of great attention. A series of mass spectrometry-based technologies have been dedicated to the characterization and quantitation of different histone forms. This review focuses on the discussion of mass spectrometry-based strategies used for the characterization of histones and their post-translational modifications. Keywordshistone; mass spectrometry; post-translational modification Histones are a group of highly conserved proteins throughout eukaryotic evolution [1]. The core histones (H4, H3, H2A and H2B) form an octamer, in which a H2A-H2B dimer associates on each side of the H3-H4 tetramer through an interaction between the C-terminal halves of H2B and H4 [2]. The negatively charged DNA superhelix wraps around the histone octamer to form repeating nucleosome cores, which further assemble into higher order chromatin fibers stabilized by the linker histones (H1 and H5) and linker DNA [3]. The histone fold regions in core histones are highly conserved α-helices, which are connected by two loops. The third histone structural motif comprises the flexible and irregular histone tails, which are extended and reach outside of the nucleosomal unit to interact with DNA [4]. Challenge of molecular characterization: post-translational modifications & sequence variationsAs a building block of the nucleosome, histones play an important role in chromatin assembly and affect the interactions between DNA and other chromatin-associated proteins. Histones regulate gene transcription through their diverse post-translational modifications (PTMs), which include lysine acetylation, lysine and arginine methylation, serine and threonine phosphorylation, lysine ubiquitination, lysine sumoylation, and poly-ADP-ribosylation of glutamic acid [5][6][7][8]. Among all the PTMs, histone acetylation and methylation have been most †

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Section: Reverse-phase High-performance Liquid Chromatography Mass Smentioning

confidence: 99%

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

Ren

Freitas

2007

Expert Review of Proteomics

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“…Over the past several years, considerable efforts have been expended to develop methods to identify the specific sites of histone modifications. Mass spectrometry (MS) coupled to liquid chromatography (LC) is the dominant technique for their characterization (7)(8)(9)(10)(11)(12)(13)(14). However, because histone proteins contain up to nearly 35% basic amino acids, the analysis of histone peptides is still problematic, as digestion with many commonly used enzymes (e.g.…”

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confidence: 99%

Comparing and Combining Capillary Electrophoresis Electrospray Ionization Mass Spectrometry and Nano–Liquid Chromatography Electrospray Ionization Mass Spectrometry for the Characterization of Post-translationally Modified Histones

Sarg

Faserl

Kremser

et al. 2013

Molecular & Cellular Proteomics

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We present the first comprehensive capillary electrophoresis electrospray ionization mass spectrometry (CESI-MS) analysis of post-translational modifications derived from H1 and core histones. Using a capillary electrophoresis system equipped with a sheathless high-sensitivity porous sprayer and nano-liquid chromatography electrospray ionization mass spectrometry (nano-LC-ESI-MS) as two complementary techniques, we characterized H1 histones isolated from rat testis. Without any pre-separation of the perchloric acid extraction, a total of 70 different modified peptides, including 50 phosphopeptides, were identified in the rat linker histones H1.0, H1a-H1e, and H1t. Out of the 70 modified H1 histone peptides, 27 peptides could be identified with CESI-MS only, and 11 solely with LC-ESI-MS. Immobilized metal-affinity chromatography enrichment prior to MS analysis yielded a total of 55 phosphopeptides; 22 of these peptides could be identified only by CESI-MS, and 19 only by LC-ESI-MS, showing the complementarity of the two techniques. We mapped 42 H1 modification sites, including 31 phosphorylation sites, of which 8 were novel sites. For the analysis of core histones, we chose a different strategy. In a first step, the sulfuric-acid-extracted core histones were pre-separated using reverse-phase high-performance liquid chromatography. Individual rat testis core histone fractions obtained in this way were digested and analyzed via bottom-up CESI-MS. This approach yielded the identification of 42 different modification sites including acetylation (lysine and N ␣ -terminal); mono-, di-, and trimethylation; and phosphorylation. When we applied CESI-MS for the analysis of intact core histone subtypes from butyrate-treated mouse tumor cells, we were able to rapidly detect their degree of modification, and we found this method very useful for the separation of isobaric trimethyl and acetyl modifications. Taken together, our results highlight the need for additional techniques for the comprehensive analysis of post-translational modifications. CESI-MS is a promising new proteomics tool as demonstrated by this, the first comprehensive analysis of histone modifications, using rat testis as an example. Molecular & Cellular

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“…A column packed with fully porous 5 μm particles, Discovery C18 (1 mm × 150 mm, 5 μm particle size, 30 nm pore size), which has been used for histone proteomics [13], was purchased from Supelco (St. Louis, MO). A column packed with superficially porous particles, Halo (1 mm × 150 mm, 3 μm particle size, 0.2 μm shell thickness, 40 nm pore size), was purchased from Advanced Materials Technology (Wilmington, DE).…”

Section: Methodsmentioning

confidence: 99%

“…With RPLC-MS, Su et al completed the profiling of whole histones and the PTMs, including methylation and acetylation [13]. Contrepois et al developed a method to characterize core histone variants and PTMs in 20 min via RPLC-LTQ-Orbitrap [14].…”

Section: Introductionmentioning

confidence: 99%

Chromatographic efficiency and selectivity in top-down proteomics of histones

Zhou¹,

Zhang²,

Fornelli

et al. 2017

Journal of Chromatography B

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Histones are involved in epigenetic control of a wide variety of cellular processes through their multiple post-translational modifications. Their strongly cationic nature makes them challenging to separate with reversed-phase liquid chromatography coupled to mass spectrometry (RPLC-MS), where trifluoroacetic acid is avoided due to adduct formation. Columns with higher resolution are needed. In this work, RPLC-MS is performed on a histone sample using difluoroacetic acid and a 20-minute gradient. Columns with C18 surfaces are compared for two different types of particle morphologies: 1) fully porous particles of 5 μm in diameter, 2) superficially porous particles of 3 μm in diameter with a shell of 0.2 μm. The resolution for the histone separation is better for the latter column, but only when the modifier is trifluoroacetic acid, which is used with UV absorbance detection. When difluoroacetic acid is used for LCMS, the peaks broaden enough to erase the advantage in efficiency for the superficially porous particles. The fully porous and superficially porous cases show similar performance in RPLC-MS, with slightly higher resolution for the fully porous particles. The expected advantage of the shorter diffusion distances for the superficially porous particles is shown to be outweighed by the lower selectivity of its bonded phase.

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Liquid chromatography mass spectrometry profiling of histones

Cited by 29 publications

References 61 publications

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

Comparing and Combining Capillary Electrophoresis Electrospray Ionization Mass Spectrometry and Nano–Liquid Chromatography Electrospray Ionization Mass Spectrometry for the Characterization of Post-translationally Modified Histones

Chromatographic efficiency and selectivity in top-down proteomics of histones

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