PRIDE Assigned Tags:Biological Dataset
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Human myogenesis is driven by proline-directed kinases and the trans-histone code, H3K9me3/H4K20me3
System-level analysis of the (phospho)proteome during muscle formation and its interplay with epigenetic factors is critical to understand muscular diseases. Using stable isotope labeling (SILAC) and nano liquid chromatography-tandem mass spectrometry (nLC-MS/MS), we analyzed the (phospho)proteome during myogenesis of LHCN-M2 human skeletal myoblast cell line. First, enriched phosphorylation motifs suggested that PKC, cyclin-dependent kinase and MAPK are regulatory kinases during myodifferentiation. Then, we uncovered that the drugs known to inhibit these kinases either promoted myogenesis (PD0325901 and GW8510) or stall differentiation (CHIR99021 and roscovitine). We identified differentiation-specific myogenic and chromatin-related proteins, including histone methyltransferases. We then analyzed histone post-translational modifications (PTMs), and observed regulation of two gene-silencing marks, H3K9me3 and H4K20me3, in a correlated manner with the observed phenotypes. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) confirmed that H3K9me3 is erased from the myogenic regulatory factors (MRFs) MyoD, MyoG and Myf5 in differentiating myotubes. Together, our work demonstrates that the integration of histone PTM, phosphoproteomics and full proteome analysis gives a comprehensive understanding of the close connection between signaling pathways and epigenetics during differentiation of myotube in vitro.
Sample Processing Protocol
The resulting peptide solution was de-salted using C18 reverse phase cartridges (Sep-Pak, Waters) and lyophilized. The dried peptides were dissolved in 5mM aqueous ammonium acetate containing 30% acetonitrile, 7mM KH2PO4, pH2.9 (solvent A) for fractionation with strong cation exchange chromatography (SCX). The SCX separation was performed on a 9.4mM x 100mM column packed with 5μM particle size, 200Å pore size SCX resin (PolySulfoethylA, PolyLC). The peptides were eluted using the following gradient program: 25% to 100% B (30% acetonitrile, 7mM KH2PO4, 350mM KCl, pH2.65) for 33 minutes followed by re-equilibration of column at 100% A. The flow rate was 2.5 mL/min, and 10 fractions were collected. Each fraction was lyophilized and re-dissolved in 0.1% acetic acid; peptides were extracted with C18 reversed phase cartridges (Oasis, Waters) prior to phosphopeptide enrichment. Phosphopeptide enrichment was performed using a TiO2 protocol described previously (Villén & Gygi, 2008). In short, about 0.5-2 mg lyophilized peptides were mixed with 500μL loading buffer (65% acetonitrile and 2% TFA saturated with glutamic acid). About 2mg TiO2 beads (Titansphere TiO2 5μm, GL Sciences Inc., Japan) were equilibrated in loading buffer for 15 min (slurry) and slurry was mixed with 0.5mg peptide(Li et al, 2009) at room temperature. TiO2 beads were then packed by centrifugation in equilibrated C18 spin columns (PepClean C18 Spin Columns, Thermo Scientific, Rockford, IL). Beads were sequentially washed with 800μL of 65% acetonitrile and 0.5% TFA followed by 65% acetonitrile and 0.1% TFA. Unbound flow-through comprised the total protein. For phosphopeptide elution, beads were incubated for 1 min in 300mM ammonium hydroxide/50% acetonitrile at room temperature and centrifuged. This sample was immediately acidified by addition of glacial acetic acid to a final concentration of 3% as phosphosites were unstable at basic pH). Two extra elution steps in 500mM ammonium hydroxide in 60% acetonitrile followed. The three eluates of each fraction were pooled, dried using a SpeedVac and pellets were stored at −80°C. Concentrated peptides were diluted with 0.1 % acetic acid for LC-MS/MS analysis.
Data Processing Protocol
MS data were analyzed using Proteome Discoverer 1.4 (Thermo Scientific) for protein identification/quantification and EpiProfile for histone quantification (Yuan et al, 2015). Mascot searches were performed against human entries in the UniProt database to assign peptide sequences and identify their proteins of origin. Precursor ion mass tolerance was set to 10 ppm; fragment ion mass tolerance was set to 0.6 Da. Enzyme was set to trypsin, and as many as two missed cleavages were allowed. Dynamic modifications included carbamidomethylation of cysteine, and oxidation of methionine, as well as phosphorylation of serine, threonine and tyrosine. SILAC labeling was set for the search, considering heavy lysines (+8 Da) and heavy arginines (+10 Da), allowing a maximum of 3 labeled amino acids (Lys and Arg) per peptide. Statistics was performed by using the two tails heteroscedastic t-test (significance when p-value <0.05); once proteins or phosphopeptide ratios were log2 transformed the t-test was used to assess whether the replicates were significantly different than 0. For histone analysis, EpiProfile performed extracted ion chromatography of a list of known modified and unmodified histone peptides. The peptide relative ratio was calculated using the total area under the extracted ion chromatograms of all peptides with the same amino acid sequence (including all of its modified forms) as 100%. For isobaric peptides, the relative ratio of two isobaric forms was estimated by averaging the ratio for each fragment ion with different mass between the two species. Statistics was assessed by using the two tails homoscedastic t-test (significant when p-value <0.05).
Zuo-Fei Yuan, UPenn
Benjamin A. Garcia, Penn Epigenetics Institute Department of Biochemistry and Biophysics Smilow Center for Translational Research University of Pennsylvania School of Medicine ( lab head )
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