Separation of acetylated core histones by hydrophilic-interaction liquid chromatography

Lindner, Herbert; Sarg, Bettina; Meraner, Christoph; Helliger, Wilfried

doi:10.1016/0021-9673(96)00131-8

Cited by 110 publications

(86 citation statements)

References 22 publications

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“…This technique is particularly suitable for separating posttranslationally modified proteins (e.g. acetylated core (45) and H1 histones (46), methylated and deamidated histones (35,(47)(48)(49), and phosphorylated H1 proteins (34, 50)). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Testis-specific Linker Histone H1t Is Multiply Phosphorylated during Spermatogenesis

Sarg

Chwatal

Talasz

et al. 2009

Journal of Biological Chemistry

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During normal spermatogenesis, the testis-specific linker histone H1t appears at pachytene stage becomes phosphorylated in early spermatids and disappears in late spermatids. Using reversed-phase and hydrophilic interaction liquid chromatography, H1t from rat and mouse testes was isolated, subjected to enzymatic digestion, and analyzed by mass spectrometry. We observed different phosphorylated states of H1t (mono-, di-, and triphosphorylated) as well as the unphosphorylated protein.Tandem mass spectrometry and immobilized metal ion affinity chromatography experiments with MS/MS/MS and multistage activation were utilized to identify five phosphorylation sites on H1t from rats. Phosphorylation occurs on both serine and threonine residues, whereas only two of these sites were located on peptides containing the CDK consensus motif (S/T)PXZ. Rat H1t phosphorylation starts first by phosphorylation of the nonconsensus motif SPKS in the COOH-terminal domain, namely at Ser-140 and to a smaller degree at a further nonconsensus motif at Ser-186. This is followed by phosphorylation of Ser-177 and Thr-155, both located in CDK consensus motifs. A single phosphorylation site at Ser-8 in the NH 2 -terminal tail was also found. Mouse H1t lacks Ser-186 and is phosphorylated at up to four sites. In contrast to somatic linker histones, no strict order of increasing phosphorylation could be detected in H1t. Thus, it appears that not the order of up-phosphorylation but the number of the phosphate groups is necessary for regulated chromatin decondensation, thus facilitating the substitution of H1t by transition proteins and protamines.Mammalian spermatogenesis is a complex, strictly organized process, including proliferation and differentiation. After several mitotic divisions of spermatogonia, germ cells differentiate into spermatocytes and pass through two consecutive meiotic divisions, resulting in haploid round cells termed spermatids. These spermatids then evolve into highly condensed and transcriptionally inert sperm through a process known as spermiogenesis (for a review, see Ref.

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Section: Resultsmentioning

confidence: 99%

Testis-specific Linker Histone H1t Is Multiply Phosphorylated during Spermatogenesis

Sarg

Chwatal

Talasz

et al. 2009

Journal of Biological Chemistry

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“…Hydrophilic-interaction LC (HILIC) is a powerful technique that has proved to be extremely well suited for histone separation. Lindner optimized HILIC to attain excellent resolution of core and linker histones [97][98][99][100]. In their work, the protein mixture was fractionated on a weak cation-exchange column using an increasing sodium perchlorate gradient in a methanephosphonic acid-triethylamine buffer (pH 3.0) in the presence of organic solvent.…”

Section: Hydrophilic-interaction Lc and Offline Hydrophilic Interactionmentioning

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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“…Bonenfant et al applied C18 RP LC-MS to the study of histones derived from Jurkat cells [16]. Hydrophilic-interaction liquid chromatography (HILIC) coupling with off-line mass spectrometry has been successful in separating and characterizing different histone isoforms [18,19]. However, the high salt required to elute the histones adds an additional desalting step prior to MS analysis.…”

Section: Introductionmentioning

confidence: 99%

Liquid chromatography mass spectrometry profiling of histones

Jacob

Amunugama

et al. 2007

Journal of Chromatography B

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Here we describe the use of reverse-phase liquid chromatography mass spectrometry (RP-LC-MS) to simultaneously characterize variants and post-translationally modified isoforms for each histone. The analysis of intact proteins significantly reduces the time of sample preparation and simplifies data interpretation. LC-MS analysis and peptide mass mapping have previously been applied to identify histone proteins and to characterize their post-translational modifications. However, these studies provided limited characterization of both linker histones and core histones. The current LC-MS analysis allows for the simultaneous observation of all histone PTMs and variants (both replacement and bulk histones) without further enrichment, which will be valuable in comparative studies. Protein identities were verified by the analysis of histone H2A species using RPLC fractionation, AU-PAGE separation and nano-LC-MS/MS.

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Separation of acetylated core histones by hydrophilic-interaction liquid chromatography

Cited by 110 publications

References 22 publications

Testis-specific Linker Histone H1t Is Multiply Phosphorylated during Spermatogenesis

Testis-specific Linker Histone H1t Is Multiply Phosphorylated during Spermatogenesis

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

Liquid chromatography mass spectrometry profiling of histones

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