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

Sidoli, Simone; Bhanu, Natarajan V.; Karch, Kelly R.; Wang, Xiaoshi; Garcia, Benjamin A.

doi:10.3791/54112

Cited by 161 publications

(183 citation statements)

References 30 publications

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“…Quantification of Histone PTMs-Histone purification and analysis was performed as previously described (31). Briefly, histones were acid extracted from nuclei with 0.2 M H 2 SO 4 for 2 h and precipitated with 33% trichloroacetic acid (TCA) for 1 h. Purified histones were dissolved in 30 l of 50 mM NH 4 HCO 3 , pH 8.0.…”

Section: Methodsmentioning

confidence: 99%

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Time-resolved Global and Chromatin Proteomics during Herpes Simplex Virus Type 1 (HSV-1) Infection

Kulej

Avgousti

Sidoli

et al. 2017

Molecular & Cellular Proteomics

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Herpes simplex virus (HSV-1) lytic infection results in global changes to the host cell proteome and the proteins associated with host chromatin. We present a system level characterization of proteome dynamics during infection by performing a multi-dimensional analysis during HSV-1 lytic infection of human foreskin fibroblast (HFF) cells. Our study includes identification and quantification of the host and viral proteomes, phosphoproteomes, chromatin bound proteomes and post-translational modifications (PTMs) on cellular histones during infection. We analyzed proteomes across six time points of virus infection (0, 3, 6, 9, 12 and 15 h post-infection) and clustered trends in abundance using fuzzy c-means. Globally, we accurately quantified more than 4000 proteins, 200 differently modified histone peptides and 9000 phosphorylation sites on cellular proteins. In addition, we identified 67 viral proteins and quantified 571 phosphorylation events (465 with high confidence site localization) on viral proteins, which is currently the most comprehensive map of HSV-1 phosphoproteome. We investigated chromatin bound proteins by proteomic analysis of the high-salt chromatin fraction and identified 510 proteins that were significantly different in abundance during infection. We found 53 histone marks significantly regulated during virus infection, including a steady increase of histone H3 acetylation (H3K9ac and H3K14ac). Our data provide a resource of unprecedented depth for human and viral proteome dynamics during infection. Collectively, our results indicate that the proteome composition of the chromatin of HFF cells is highly affected during HSV-1 infection, and that phosphorylation events are abundant on viral proteins. We propose that our epi-proteomics approach will prove to be important in the characterization of other model infectious systems that involve changes to chromatin composition. Molecular & Cellular Proteomics 16: 10.1074/mcp.M116.065987, S92-S107, 2017. Herpes simplex virus (HSV-1)1 leads to a contagious and persistent infection that affects about 95% of the human population. The HSV-1 genome is a double-stranded DNA molecule that replicates in the host cell nucleus (1). HSV-1 initially infects epithelial cells as a lytic infection, and then enters peripheral neurons where it establishes latency (2, 3). Processes such as viral transcription, viral DNA synthesis, virion assembly and DNA packaging take place in discrete virus-induced structures within the nucleus called replication compartments (1, 4). These processes are temporally regulated by the viral cascades of immediate-early (IE), early (E), and late (L) gene expression. The IE proteins are primarily responsible for counteracting intrinsic host defenses and for activating expression of early-phase genes (1). Early viral pro-

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Section: Methodsmentioning

confidence: 99%

“…To isolate histones we performed purification via acidic extraction, and followed this by derivatization with propionic anhydride (31). After digestion, we performed dataindependent acquisition in MS to quantify accurately the canonical and isobaric peptides (95,96).…”

Section: Phosphoproteomics Analysis Of Host and Viral Proteome Duringmentioning

confidence: 99%

Time-resolved Global and Chromatin Proteomics during Herpes Simplex Virus Type 1 (HSV-1) Infection

Kulej

Avgousti

Sidoli

et al. 2017

Molecular & Cellular Proteomics

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“…Reactions were stopped by freezing, and samples were dried in a SpeedVac and resuspended in 20 μl of 100 mM ammonium bicarbonate. Histones were derivatized and analyzed by mass spectrometry following procedures described previously (Sidoli et al, 2016). Data analysis was performed using the software EpiProfile with a 10 ppm tolerance for extracting peak areas from raw files (Yuan et al, 2015).…”

Section: Star Methodsmentioning

confidence: 99%

Recognition of Histone H3K14 Acylation by MORF

et al. 2017

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SUMMARY The monocytic leukemia zinc-finger protein-related factor (MORF) is a transcriptional coactivator and a catalytic subunit of the lysine acetyltransferase complex implicated in cancer and developmental diseases. We have previously shown that the double plant homeodomain finger (DPF) of MORF is capable of binding to acetylated histone H3. Here we demonstrate that the DPF of MORF recognizes many newly identified acylation marks. The mass spectrometry study provides comprehensive analysis of H3K14 acylation states in vitro and in vivo. The crystal structure of the MORF DPF-H3K14butyryl complex offers insight into the selectivity of this reader toward lipophilic acyllysine substrates. Together, our findings support the mechanism by which the acetyltransferase MORF promotes spreading of histone acylation.

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“…Histones were then extracted using a standard sulfuric acid extraction followed by TCA precipitation as previously described. 28–30 For all experiments where in nucleo incubation was not performed, nuclei were extracted from fresh or frozen pellets as previously described using detergent followed by an acid extraction and TCA precipitation. 28–30 …”

Section: Methodsmentioning

confidence: 99%

“…Samples were then desalted on home-made stage tip columns with C18 solid phase extraction resin (3M, Empore) and dried to completion in a vacuum centrifuge as previously described. 28,29 …”

Section: Methodsmentioning

confidence: 99%

The nucleosomal surface is the main target of histone ADP-ribosylation in response to DNA damage

et al. 2017

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ADP-ribosylation is a protein post-translational modification catalyzed by ADP-ribose transferases (ARTs). ART activity is critical in mediating many cellular processes, and is required for DNA damage repair. All five histone proteins are extensively ADP-ribosylated by ARTs upon induction of DNA damage. However, how these modifications aid in repair processes is largely unknown, primarily due to lack of knowledge about where they site-specifically occur on histones. Here, we conduct a comprehensive analysis of histone Asp/Glu ADP-ribosylation sites upon DNA damage induced by dimethyl sulfate (DMS). We also demonstrate that incubation of cell nuclei with NAD+, as has been done previously in the literature, leads to spurious ADP-ribosylation levels of histone proteins. Altogether, we were able to identify 30 modification sites, 20 of which are novel. We also quantify the abundance of these modification sites during the course of DNA damage insult to identify which sites are critical for mediating repair. We found that every quantifiable site increases in abundance over time and that each identified ADP-ribosylation site is located on the surface of the nucleosome. Together, the data suggest specific Asp/Glu residues are unlikely to be critical for DNA damage repair and rather that this process is likely dependent on ADP-ribosylation of the nucleosomal surface in general.

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

Cited by 161 publications

References 30 publications

Time-resolved Global and Chromatin Proteomics during Herpes Simplex Virus Type 1 (HSV-1) Infection

Time-resolved Global and Chromatin Proteomics during Herpes Simplex Virus Type 1 (HSV-1) Infection

Recognition of Histone H3K14 Acylation by MORF

The nucleosomal surface is the main target of histone ADP-ribosylation in response to DNA damage

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