In-gel NHS-propionate derivatization for histone post-translational modifications analysis in Arabidopsis thaliana

Chen, Jiajia; Gao, Jun; Peng, Maolin; Wang, Yi; Yu, Yanyan; Yang, Pengyuan; Jin, Hong

doi:10.1016/j.aca.2015.06.019

Cited by 7 publications

(9 citation statements)

References 22 publications

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“…Then, the peptides were directly analysed online using QTRAP 6500 mass spectrometer (Applied Biosystems, Singapore) set to multiple reaction monitoring (MRM) in positive electrospray ionization mode. The mass transitions were selected as previously described by Chen et al 54 55 , with the addition of transitions corresponding to 13 C 2 -labelled acetylated peptides. All MRM data were processed through Skyline software (Washington University in St Louis, St Louis, MO, USA).…”

Section: Methodsmentioning

confidence: 99%

Acetate functions as an epigenetic metabolite to promote lipid synthesis under hypoxia

et al. 2016

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Besides the conventional carbon sources, acetyl-CoA has recently been shown to be generated from acetate in various types of cancers, where it promotes lipid synthesis and tumour growth. The underlying mechanism, however, remains largely unknown. We find that acetate induces a hyperacetylated state of histone H3 in hypoxic cells. Acetate predominately activates lipogenic genes ACACA and FASN expression by increasing H3K9, H3K27 and H3K56 acetylation levels at their promoter regions, thus enhancing de novo lipid synthesis, which combines with its function as the metabolic precursor for fatty acid synthesis. Acetyl-CoA synthetases (ACSS1, ACSS2) are involved in this acetate-mediated epigenetic regulation. More importantly, human hepatocellular carcinoma with high ACSS1/2 expression exhibit increased histone H3 acetylation and FASN expression. Taken together, this study demonstrates that acetate, in addition to its ability to induce fatty acid synthesis as an immediate metabolic precursor, also functions as an epigenetic metabolite to promote cancer cell survival under hypoxic stress.

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Section: Methodsmentioning

confidence: 99%

Acetate functions as an epigenetic metabolite to promote lipid synthesis under hypoxia

et al. 2016

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“…To investigate histone modification states, various strategies based on immunological procedures or mass spectrometry (MS) are available. While ChIP-based methods (e.g., ChIP-seq or ChIP-on-chip) are favored for analysis of histone marks at functionally distinct chromatin ( Ha et al, 2011 ; Roudier et al, 2011 ; Sequeira-Mendes et al, 2014 ; Zhang et al, 2015 ), MS-based analysis is better suited to study of a system-level view of histone modifications ( Johnson et al, 2004 ; Chen et al, 2015 ; Moraes et al, 2015 ). In addition to the identification and quantification of histone marks, unlike specific antibodies MS enables detection of novel hPTMs ( Strahl et al, 2001 ; Gardner et al, 2011 ).…”

Section: Introductionmentioning

confidence: 99%

“…Improvements in retention of hydrophilic peptides, e.g., H3K4me2/me3, and recoveries have been achieved by hybrid histone labeling using propionic anhydride for protein derivatization followed by post-digestion labeling of peptide N-termini using phenylisocyanate (PIC; Maile et al, 2015 ). In-gel variants of propionylation have also been developed, based on either a standard protocol using propionic anhydride or NHS-propionate reagent ( Ouvry-Patat and Schey, 2007 ; Chen et al, 2015 ).…”

Section: Introductionmentioning

confidence: 99%

“…Difficulties encountered during plant histone sample preparation for MS are reflected by the limited number of studies focused on plant histone characterization. Moreover, only a few studies dealing with plant hPTM used the advantage of shotgun proteomics coupled with lysine derivatization which is widely used for mammalian histone analyses ( Johnson et al, 2004 ; Chen et al, 2015 ; Moraes et al, 2015 ). Johnson’s and Chen’s groups performed prefractionation of histone samples isolated from Arabidopsis leaves prior propionylation of histone H3.…”

Section: Introductionmentioning

confidence: 99%

“…Johnson’s and Chen’s groups performed prefractionation of histone samples isolated from Arabidopsis leaves prior propionylation of histone H3. While Johnson et al (2004) purified histone H3 N-termini from dialyzed crude histone extract using double-round HPLC, Chen et al (2015) separated histone sample after acidic extraction using SDS–PAGE and performed NHS-propionate derivatization in gel pieces corresponding to histone H3. Moraes et al (2015) omitted histone fractionation and used dialyzed total histone crude extract from sugarcane for propionylation to characterize PTMs at histone H3 and H4 by LC-MS/MS.…”

Section: Introductionmentioning

confidence: 99%

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Filter-Aided Sample Preparation Procedure for Mass Spectrometric Analysis of Plant Histones

et al. 2018

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Characterization of histone post-translational modifications (PTMs) is still challenging, and robust histone sample preparation is essential for convincing evaluation of PTMs by mass spectrometry. An effective protocol for extracting plant histone proteins must also avoid excessive co-extraction of the numerous potential interfering compounds, including those related to secondary metabolism. Currently, the co-existence of histone marks is addressed mostly by shotgun proteomic analysis following chemical derivatization of histone lysine residues. Here, we report a straightforward approach for plant histone sample preparation for mass spectrometry, based on filter-aided sample preparation coupled with histone propionylation. The approach offers savings in sample handling and preparation time, enables removal of interfering compounds from the sample, and does not require either precipitation or dialysis of histone extract. We show the comparison of two protocol variants for derivatization of histone proteins, in-solution propionylation in the vial and propionylation on the filter unit. For both protocols, we obtained identical abundances of post-translationally modified histone peptides. Although shorter time is required for histone protein labeling on the filter unit, in-solution derivatization slightly outweighed filter-based variant by lower data variability. Nevertheless, both protocol variants appear to be efficient and convenient approach for preparation of plant histones for mass spectrometric analysis.

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Histone Acid Extraction and High Throughput Mass Spectrometry to Profile Histone Modifications in Arabidopsis thaliana

et al. 2022

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Histone post-translational modifications (PTMs) play important roles in many biological processes, including gene regulation and chromatin dynamics, and are thus of high interest across many fields of biological research. Chromatin immunoprecipitation coupled with sequencing (ChIP-seq) is a powerful tool to profile histone PTMs in vivo. This method, however, is largely dependent on the specificity and availability of suitable commercial antibodies. While mass spectrometry (MS)-based proteomic approaches to quantitatively measure histone PTMs have been developed in mammals and several other model organisms, such methods are currently not readily available in plants. One major challenge for the implementation of such methods in plants has been the difficulty in isolating sufficient amounts of pure, high-quality histones, a step rendered difficult by the presence of the cell wall. Here, we developed a high-yielding histone extraction and purification method optimized for Arabidopsis thaliana that can be used to obtain high-quality histones for MS. In contrast to other methods used in plants, this approach is relatively simple, and does not require membranes or additional specialized steps, such as gel excision or chromatography, to extract highly purified histones. We also describe methods for producing MS-ready histone peptides through chemical labeling and digestion. Finally, we describe an optimized method to quantify and analyze the resulting histone PTM data using a modified version of EpiProfile 2.0 for Arabidopsis. In all, the workflow described here can be used to measure changes to histone PTMs resulting from various treatments, stresses, and time courses, as well as in different mutant lines.

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In-gel NHS-propionate derivatization for histone post-translational modifications analysis in Arabidopsis thaliana

Cited by 7 publications

References 22 publications

Acetate functions as an epigenetic metabolite to promote lipid synthesis under hypoxia

Acetate functions as an epigenetic metabolite to promote lipid synthesis under hypoxia

Filter-Aided Sample Preparation Procedure for Mass Spectrometric Analysis of Plant Histones

Histone Acid Extraction and High Throughput Mass Spectrometry to Profile Histone Modifications in Arabidopsis thaliana

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