EpiProfile Quantifies Histone Peptides With Modifications by Extracting Retention Time and Intensity in High-resolution Mass Spectra*

Yuan, Zuo Fei; Lin, Shu; Molden, Rosalynn C.; Cao, Xing Jun; Bhanu, Natarajan V.; Wang, Xiaoshi; Sidoli, Simone; Liu, Shichong; Garcia, Benjamin A.

doi:10.1074/mcp.m114.046011

Cited by 88 publications

(114 citation statements)

References 28 publications

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“…After digestion, we performed dataindependent acquisition in MS to quantify accurately the canonical and isobaric peptides (95,96). Chromatographic peak area extraction was performed using the software EpiProfile (32). We quantified 140 different histone peptides ( Fig.…”

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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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: 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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“…As illustrated previously (65), we targeted the m/z for the isobaric peptides and quantified the relative abundance of their unique b or y ions at the MS/MS level. Subsequently, we determined the relative abundance at the MS1 level based on the ratios we obtained, either manually or by a Matlab-based program, EpiProfile (69). Data were corrected for differences in detection efficiencies based on the correction factors generated by a synthetic peptide library as described previously (70).…”

Section: Nano-liquid Chromatography Electrospray Ionization Tandem Mamentioning

confidence: 99%

Preferential Phosphorylation on Old Histones during Early Mitosis in Human Cells

Lin

Yuan

Han

et al. 2016

Journal of Biological Chemistry

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How histone post-translational modifications (PTMs) are inherited through the cell cycle remains poorly understood. Canonical histones are made in the S phase of the cell cycle. Combining mass spectrometry-based technologies and stable isotope labeling by amino acids in cell culture, we question the distribution of multiple histone PTMs on old versus new histones in synchronized human cells. We show that histone PTMs can be grouped into three categories according to their distributions. Most lysine mono-methylation and acetylation PTMs are either symmetrically distributed on old and new histones or are enriched on new histones. In contrast, most di-and tri-methylation PTMs are enriched on old histones, suggesting that the inheritance of different PTMs is regulated distinctly. Intriguingly, old and new histones are distinct in their phosphorylation status during early mitosis in the following three human cell types: HeLa, 293T, and human foreskin fibroblast cells. The mitotic hallmark H3S10ph is predominantly associated with old H3 at early mitosis and becomes symmetric with the progression of mitosis. This same distribution was observed with other mitotic phosphorylation marks, including H3T3/T6ph, H3.1/ 2S28ph, and H1.4S26ph but not S28/S31ph on the H3 variant H3.3. Although H3S10ph often associates with the neighboring Lys-9 di-or tri-methylations, they are not required for the asymmetric distribution of Ser-10 phosphorylation on the same H3 tail. Inhibition of the kinase Aurora B does not change the distribution despite significant reduction of H3S10ph levels. However, K9me2 abundance on the new H3 is significantly reduced after Aurora B inhibition, suggesting a cross-talk between H3S10ph and H3K9me2.In eukaryotes, histone proteins facilitate the packaging of DNA molecules. The DNA double helix wraps around histone octamers to form nucleosomes. A histone octamer contains two copies of each core histone H3, H4, H2A, and H2B. A 5th histone, histone H1, is associated with the linker DNA which lies between the nucleosomes. Canonical histone proteins are cell cycle-dependent and are produced in S phase (1, 2), whereas cell cycle-independent histone variants (e.g. H3.3) are synthesized throughout the cell cycle (3). Histone proteins carry numerous post-translational modifications (PTMs) 3 that are involved in multiple functions such as epigenetic regulation of transcription, DNA damage repair, and cell cycle progression (4, 5). To maintain lineage identity and to guide proper transcription, cells must replicate PTMs from old histones onto new histones at each cell division. Major efforts have been devoted to understanding how histones themselves are transmitted through the DNA replication fork in S phase (6). In principle, the newly deposited nucleosomes could contain entirely old or newly synthesized histone proteins, or a mixture of both. Accumulating evidence suggests that most H3/H4 tetramers remain intact, with the exception of some H3.3/H4 tetramers, indicating that nucleosomes should contain either new or ...

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“…Raw MS data were analyzed adopting software for the extraction of precursor and fragment ion chromatograms, namely Skyline 23 and EpiProfile 22 . EpiProfile has been optimized for histone peptides, as it integrates intelligent peak area extraction due to previous knowledge of peptide retention time.…”

Section: Representative Resultsmentioning

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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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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EpiProfile Quantifies Histone Peptides With Modifications by Extracting Retention Time and Intensity in High-resolution Mass Spectra*

Cited by 88 publications

References 28 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

Preferential Phosphorylation on Old Histones during Early Mitosis in Human Cells

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

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