5 research outputs found

    High-Throughput Protein Sequencing

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    We have designed and implemented an autosampler that provides additional sample capacity on a commercial protein sequencer. The autosampler attaches to a standard ABI Procise sequencer, enabling a single-sample cartridge to hold up to six separate samples. The autosampler is used in combination with faster Edman cycles and a rapid 12-min PTH separation to significantly increase the speed of automated protein sequencing. We also describe Edman chemistry modifications that improve coupling efficiency and prevent internal cleavage that can occur when samples are sequenced in the standard glass cartridges

    Supplementary Information from Ras–MEK Signaling Mediates a Critical Chk1-Dependent DNA Damage Response in Cancer Cells

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    Supplementary Methods Supplementary Figure S1: Gö6976 selectively inhibits osteosarcoma cell growth through Chk1 inhibition in a p53-independent manner Supplementary Figure S2: Relationship between GDC-0425 sensitivity and Chk1 expression in cell lines derived from the indicated tissue types Supplementary Figure S3: Ras-MEK signaling is associated with Chk1 dependency Supplementary Figure S4: Feedback activation of Ras-MEK by Chk1 inhibition Supplementary Figure S5: BT-549 cells were treated with either GDC-0425 or cobimetinib alone or combination for 24 hr. Supplementary Figure S6: Synergistic anti-tumor growth activity by gemcitabine/GDC-0425 combination treatment in Chk1-dependent cancer cells.</p

    Development of Orthogonally Protected Hypusine for Solid-Phase Peptide Synthesis

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    An orthogonally protected hypusine reagent was developed for solid-phase synthesis of hypusinated peptides using the Fmoc/<i>t</i>-Bu protection strategy. The reagent was synthesized in an overall yield of 27% after seven steps from Cbz-Lys-OBzl and (<i>R</i>)-3-hydroxypyrrolidin-2-one. The side-chain protecting groups (Boc and <i>t</i>-Bu) are fully compatible with standard Fmoc chemistry and can be readily removed during the peptide cleavage step. The utility of the reagent was demonstrated by solid-phase synthesis of hypusinated peptides

    Proteomic Analyses Identify a Novel Role for EZH2 in the Initiation of Cancer Cell Drug Tolerance

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    Acquisition of drug resistance remains a chief impediment to successful cancer therapy, and we previously described a transient drug-tolerant cancer cell population (DTPs) whose survival is in part dependent on the activities of the histone methyltransferases G9a/EHMT2 and EZH2, the latter being the catalytic component of the polycomb repressive complex 2 (PRC2). Here, we apply multiple proteomic techniques to better understand the role of these histone methyltransferases (HMTs) in the establishment of the DTP state. Proteome-wide comparisons of lysine methylation patterns reveal that DTPs display an increase in methylation on K116 of PRC member Jarid2, an event that helps stabilize and recruit PRC2 to chromatin. We also find that EZH2, in addition to methylating histone H3K27, also can methylate G9a at K185, and that methylated G9a better recruits repressive complexes to chromatin. These complexes are similar to complexes recruited by histone H3 methylated at K9. Finally, a detailed histone post-translational modification (PTM) analysis shows that EZH2, either directly or through its ability to methylate G9a, alters H3K9 methylation in the context of H3 serine 10 phosphorylation, primarily in a cancer cell subpopulation that serves as DTP precursors. We also show that combinations of histone PTMs recruit a different set of complexes to chromatin, shedding light on the temporal mechanisms that contribute to drug tolerance
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