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Everley, Robert A

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Everley

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Robert A

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Everley, Robert A
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    Evaluating Multiplexed Quantitative Phosphopeptide Analysis on a Hybrid Quadrupole Mass Filter/Linear Ion Trap/Orbitrap Mass Spectrometer
    (American Chemical Society, 2014) Erickson, Brian K.; Jedrychowski, Mark; McAlister, Graeme C.; Everley, Robert A; Kunz, Ryan; Gygi, Steven
    As a driver for many biological processes, phosphorylation remains an area of intense research interest. Advances in multiplexed quantitation utilizing isobaric tags (e.g., TMT and iTRAQ) have the potential to create a new paradigm in quantitative proteomics. New instrumentation and software are propelling these multiplexed workflows forward, which results in more accurate, sensitive, and reproducible quantitation across tens of thousands of phosphopeptides. This study assesses the performance of multiplexed quantitative phosphoproteomics on the Orbitrap Fusion mass spectrometer. Utilizing a two-phosphoproteome model of precursor ion interference, we assessed the accuracy of phosphopeptide quantitation across a variety of experimental approaches. These methods included the use of synchronous precursor selection (SPS) to enhance TMT reporter ion intensity and accuracy. We found that (i) ratio distortion remained a problem for phosphopeptide analysis in multiplexed quantitative workflows, (ii) ratio distortion can be overcome by the use of an SPS-MS3 scan, (iii) interfering ions generally possessed a different charge state than the target precursor, and (iv) selecting only the phosphate neutral loss peak (single notch) for the MS3 scan still provided accurate ratio measurements. Remarkably, these data suggest that the underlying cause of interference may not be due to coeluting and cofragmented peptides but instead from consistent, low level background fragmentation. Finally, as a proof-of-concept 10-plex experiment, we compared phosphopeptide levels from five murine brains to five livers. In total, the SPS-MS3 method quantified 38 247 phosphopeptides, corresponding to 11 000 phosphorylation sites. With 10 measurements recorded for each phosphopeptide, this equates to more than 628 000 binary comparisons collected in less than 48 h.
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    Regulation of Selenocysteine Content of Human Selenoprotein P by Dietary Selenium and Insertion of Cysteine in Place of Selenocysteine
    (Public Library of Science, 2015) Turanov, Anton A.; Everley, Robert A; Hybsier, Sandra; Renko, Kostja; Schomburg, Lutz; Gygi, Steven; Hatfield, Dolph L.; Gladyshev, Vadim
    Selenoproteins are a unique group of proteins that contain selenium in the form of selenocysteine (Sec) co-translationally inserted in response to a UGA codon with the help of cis- and trans-acting factors. Mammalian selenoproteins contain single Sec residues, with the exception of selenoprotein P (SelP) that has 7–15 Sec residues depending on species. Assessing an individual’s selenium status is important under various pathological conditions, which requires a reliable selenium biomarker. Due to a key role in organismal selenium homeostasis, high Sec content, regulation by dietary selenium, and availability of robust assays in human plasma, SelP has emerged as a major biomarker of selenium status. Here, we found that Cys is present in various Sec positions in human SelP. Treatment of cells expressing SelP with thiophosphate, an analog of the selenium donor for Sec synthesis, led to a nearly complete replacement of Sec with Cys, whereas supplementation of cells with selenium supported Sec insertion. SelP isolated directly from human plasma had up to 8% Cys inserted in place of Sec, depending on the Sec position. These findings suggest that a change in selenium status may be reflected in both SelP concentration and its Sec content, and that availability of the SelP-derived selenium for selenoprotein synthesis may be overestimated under conditions of low selenium status due to replacement of Sec with Cys.
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    Combining Amine Metabolomics and Quantitative Proteomics of Cancer Cells Using Derivatization with Isobaric Tags
    (American Chemical Society, 2014) Murphy, J. Patrick; Everley, Robert A; Coloff, Jonathan L.; Gygi, Steven
    Quantitative metabolomics and proteomics technologies are powerful approaches to explore cellular metabolic regulation. Unfortunately, combining the two technologies typically requires different LC-MS setups for sensitive measurement of metabolites and peptides. One approach to enhance the analysis of certain classes of metabolites is by derivatization with various types of tags to increase ionization and chromatographic efficiency. We demonstrate here that derivatization of amine metabolites with tandem mass tags (TMT), typically used in multiplexed peptide quantitation, facilitates amino acid analysis by standard nanoflow reversed-phase LC-MS setups used for proteomics. We demonstrate that this approach offers the potential to perform experiments at the MS1-level using duplex tags or at the MS2-level using novel 10-plex reporter ion-containing isobaric tags for multiplexed amine metabolite analysis. We also demonstrate absolute quantitative measurements of amino acids conducted in parallel with multiplexed quantitative proteomics, using similar LC-MS setups to explore cellular amino acid regulation. We further show that the approach can also be used to determine intracellular metabolic labeling of amino acids from glucose carbons.
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    Comparative Analysis of Erk Phosphorylation Suggests a Mixed Strategy for Measuring Phospho-Form Distributions
    (Nature Publishing Group, 2011) Prabakaran, Sudhakaran; Everley, Robert A; Landrieu, Isabelle; Wieruszeski, Jean-Michel; Lippens, Guy; Steen, Hanno; Gunawardena, Jeremy
    The functional impact of multisite protein phosphorylation can depend on both the numbers and the positions of phosphorylated sites—the global pattern of phosphorylation or ‘phospho-form’—giving biological systems profound capabilities for dynamic information processing. A central problem in quantitative systems biology, therefore, is to measure the ‘phospho-form distribution’: the relative amount of each of the 2\(^n\) phospho-forms of a protein with n-phosphorylation sites. We compared four potential methods—western blots with phospho-specific antibodies, peptide-based liquid chromatography (LC) and mass spectrometry (MS; pepMS), protein-based LC/MS (proMS) and nuclear magnetic resonance spectroscopy (NMR)—on differentially phosphorylated samples of the well-studied mitogen-activated protein kinase Erk2, with two phosphorylation sites. The MS methods were quantitatively consistent with each other and with NMR to within 10%, but western blots, while highly sensitive, showed significant discrepancies with MS. NMR also uncovered two additional phosphorylations, for which a combination of pepMS and proMS yielded an estimate of the 16-member phospho-form distribution. This combined MS strategy provides an optimal mixture of accuracy and coverage for quantifying distributions, but positional isomers remain a challenging problem.