Publications by authors named "Robert Everley"

33 Publications

The RESOLUTE consortium: unlocking SLC transporters for drug discovery.

Authors:
Giulio Superti-Furga Daniel Lackner Tabea Wiedmer Alvaro Ingles-Prieto Barbara Barbosa Enrico Girardi Ulrich Goldmann Bettina Gürtl Kristaps Klavins Christoph Klimek Sabrina Lindinger Eva Liñeiro-Retes André C Müller Svenja Onstein Gregor Redinger Daniela Reil Vitaly Sedlyarov Gernot Wolf Matthew Crawford Robert Everley David Hepworth Shenping Liu Stephen Noell Mary Piotrowski Robert Stanton Hui Zhang Salvatore Corallino Andrea Faedo Maria Insidioso Giovanna Maresca Loredana Redaelli Francesca Sassone Lia Scarabottolo Michela Stucchi Paola Tarroni Sara Tremolada Helena Batoulis Andreas Becker Eckhard Bender Yung-Ning Chang Alexander Ehrmann Anke Müller-Fahrnow Vera Pütter Diana Zindel Bradford Hamilton Martin Lenter Diana Santacruz Coralie Viollet Charles Whitehurst Kai Johnsson Philipp Leippe Birgit Baumgarten Lena Chang Yvonne Ibig Martin Pfeifer Jürgen Reinhardt Julian Schönbett Paul Selzer Klaus Seuwen Charles Bettembourg Bruno Biton Jörg Czech Hélène de Foucauld Michel Didier Thomas Licher Vincent Mikol Antje Pommereau Frédéric Puech Veeranagouda Yaligara Aled Edwards Brandon J Bongers Laura H Heitman Ad P IJzerman Huub J Sijben Gerard J P van Westen Justine Grixti Douglas B Kell Farah Mughal Neil Swainston Marina Wright-Muelas Tina Bohstedt Nicola Burgess-Brown Liz Carpenter Katharina Dürr Jesper Hansen Andreea Scacioc Giulia Banci Claire Colas Daniela Digles Gerhard Ecker Barbara Füzi Viktoria Gamsjäger Melanie Grandits Riccardo Martini Florentina Troger Patrick Altermatt Cédric Doucerain Franz Dürrenberger Vania Manolova Anna-Lena Steck Hanna Sundström Maria Wilhelm Claire M Steppan

Nat Rev Drug Discov 2020 07;19(7):429-430

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http://dx.doi.org/10.1038/d41573-020-00056-6DOI Listing
July 2020

Multi omics analysis of fibrotic kidneys in two mouse models.

Sci Data 2019 06 14;6(1):92. Epub 2019 Jun 14.

Laboratory of Systems Pharmacology, Harvard Medical School, Boston, MA, USA.

Kidney fibrosis represents an urgent unmet clinical need due to the lack of effective therapies and an inadequate understanding of the molecular pathogenesis. We have generated a comprehensive and combined multi-omics dataset (proteomics, mRNA and small RNA transcriptomics) of fibrotic kidneys that is searchable through a user-friendly web application: http://hbcreports.med.harvard.edu/fmm/ . Two commonly used mouse models were utilized: a reversible chemical-induced injury model (folic acid (FA) induced nephropathy) and an irreversible surgically-induced fibrosis model (unilateral ureteral obstruction (UUO)). mRNA and small RNA sequencing, as well as 10-plex tandem mass tag (TMT) proteomics were performed with kidney samples from different time points over the course of fibrosis development. The bioinformatics workflow used to process, technically validate, and combine the single omics data will be described. In summary, we present temporal multi-omics data from fibrotic mouse kidneys that are accessible through an interrogation tool (Mouse Kidney Fibromics browser) to provide a searchable transcriptome and proteome for kidney fibrosis researchers.
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http://dx.doi.org/10.1038/s41597-019-0095-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6570759PMC
June 2019

Multiomics Profiling Establishes the Polypharmacology of FDA-Approved CDK4/6 Inhibitors and the Potential for Differential Clinical Activity.

Cell Chem Biol 2019 08 6;26(8):1067-1080.e8. Epub 2019 Jun 6.

Laboratory of Systems Pharmacology, Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

The target profiles of many drugs are established early in their development and are not systematically revisited at the time of FDA approval. Thus, it is often unclear whether therapeutics with the same nominal targets but different chemical structures are functionally equivalent. In this paper we use five different phenotypic and biochemical assays to compare approved inhibitors of cyclin-dependent kinases 4/6-collectively regarded as breakthroughs in the treatment of hormone receptor-positive breast cancer. We find that transcriptional, proteomic, and phenotypic changes induced by palbociclib, ribociclib, and abemaciclib differ significantly; abemaciclib in particular has advantageous activities partially overlapping those of alvocidib, an older polyselective CDK inhibitor. In cells and mice, abemaciclib inhibits kinases other than CDK4/6 including CDK2/cyclin A/E-implicated in resistance to CDK4/6 inhibition-and CDK1/cyclin B. The multifaceted experimental and computational approaches described here therefore uncover underappreciated differences in CDK4/6 inhibitor activities with potential importance in treating human patients.
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http://dx.doi.org/10.1016/j.chembiol.2019.05.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6936329PMC
August 2019

PF-06651600, a Dual JAK3/TEC Family Kinase Inhibitor.

ACS Chem Biol 2019 06 22;14(6):1235-1242. Epub 2019 May 22.

Inflammation and Immunology , Pfizer Worldwide R&D , 610 Main Street , Cambridge , Massachusetts 02139 , United States.

PF-06651600 was developed as an irreversible inhibitor of JAK3 with selectivity over the other three JAK isoforms. A high level of selectivity toward JAK3 is achieved by the covalent interaction of PF-06651600 with a unique cysteine residue (Cys-909) in the catalytic domain of JAK3, which is replaced by a serine residue in the other JAK isoforms. Importantly, 10 other kinases in the kinome have a cysteine at the equivalent position of Cys-909 in JAK3. Five of those kinases belong to the TEC kinase family including BTK, BMX, ITK, RLK, and TEC and are also inhibited by PF-06651600. Preclinical data demonstrate that inhibition of the cytolytic function of CD8 T cells and NK cells by PF-06651600 is driven by the inhibition of TEC kinases. On the basis of the underlying pathophysiology of inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, alopecia areata, and vitiligo, the dual activity of PF-06651600 toward JAK3 and the TEC kinase family may provide a beneficial inhibitory profile for therapeutic intervention.
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http://dx.doi.org/10.1021/acschembio.9b00188DOI Listing
June 2019

Adaptation of Human iPSC-Derived Cardiomyocytes to Tyrosine Kinase Inhibitors Reduces Acute Cardiotoxicity via Metabolic Reprogramming.

Cell Syst 2019 05 8;8(5):412-426.e7. Epub 2019 May 8.

Laboratory of Systems Pharmacology, Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Tyrosine kinase inhibitors (TKIs) are widely used to treat solid tumors but can be cardiotoxic. The molecular basis for this toxicity and its relationship to therapeutic mechanisms remain unclear; we therefore undertook a systems-level analysis of human cardiomyocytes (CMs) exposed to four TKIs. CMs differentiated from human induced pluripotent stem cells (hiPSCs) were exposed to sunitinib, sorafenib, lapatinib, or erlotinib, and responses were assessed by functional assays, microscopy, RNA sequencing, and mass spectrometry (GEO: GSE114686; PRIDE: PXD012043). TKIs have diverse effects on hiPSC-CMs distinct from inhibition of tyrosine-kinase-mediated signal transduction; cardiac metabolism is particularly sensitive. Following sorafenib treatment, oxidative phosphorylation is downregulated, resulting in a profound defect in mitochondrial energetics. Cells adapt by upregulating aerobic glycolysis. Adaptation makes cells less acutely sensitive to sorafenib but may have long-term negative consequences. Thus, CMs exhibit adaptive responses to anti-cancer drugs conceptually similar to those previously shown in tumors to mediate drug resistance.
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http://dx.doi.org/10.1016/j.cels.2019.03.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6657491PMC
May 2019

Dual Sensing of Physiologic pH and Calcium by EFCAB9 Regulates Sperm Motility.

Cell 2019 05 2;177(6):1480-1494.e19. Epub 2019 May 2.

Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT 06510, USA; Department of Gynecology and Obstetrics, Yale School of Medicine, New Haven, CT 06510, USA. Electronic address:

Varying pH of luminal fluid along the female reproductive tract is a physiological cue that modulates sperm motility. CatSper is a sperm-specific, pH-sensitive calcium channel essential for hyperactivated motility and male fertility. Multi-subunit CatSper channel complexes organize linear Ca signaling nanodomains along the sperm tail. Here, we identify EF-hand calcium-binding domain-containing protein 9 (EFCAB9) as a bifunctional, cytoplasmic machine modulating the channel activity and the domain organization of CatSper. Knockout mice studies demonstrate that EFCAB9, in complex with the CatSper subunit, CATSPERζ, is essential for pH-dependent and Ca-sensitive activation of the CatSper channel. In the absence of EFCAB9, sperm motility and fertility is compromised, and the linear arrangement of the Ca signaling domains is disrupted. EFCAB9 interacts directly with CATSPERζ in a Ca-dependent manner and dissociates at elevated pH. These observations suggest that EFCAB9 is a long-sought, intracellular, pH-dependent Ca sensor that triggers changes in sperm motility.
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http://dx.doi.org/10.1016/j.cell.2019.03.047DOI Listing
May 2019

Leveraging Compound Promiscuity to Identify Targetable Cysteines within the Kinome.

Cell Chem Biol 2019 06 11;26(6):818-829.e9. Epub 2019 Apr 11.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Covalent kinase inhibitors, which typically target cysteine residues, represent an important class of clinically relevant compounds. Approximately 215 kinases are known to have potentially targetable cysteines distributed across 18 spatially distinct locations proximal to the ATP-binding pocket. However, only 40 kinases have been covalently targeted, with certain cysteine sites being the primary focus. To address this disparity, we have developed a strategy that combines the use of a multi-targeted acrylamide-modified inhibitor, SM1-71, with a suite of complementary chemoproteomic and cellular approaches to identify additional targetable cysteines. Using this single multi-targeted compound, we successfully identified 23 kinases that are amenable to covalent inhibition including MKNK2, MAP2K1/2/3/4/6/7, GAK, AAK1, BMP2K, MAP3K7, MAPKAPK5, GSK3A/B, MAPK1/3, SRC, YES1, FGFR1, ZAK (MLTK), MAP3K1, LIMK1, and RSK2. The identification of nine of these kinases previously not targeted by a covalent inhibitor increases the number of targetable kinases and highlights opportunities for covalent kinase inhibitor development.
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http://dx.doi.org/10.1016/j.chembiol.2019.02.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6634314PMC
June 2019

A dynamic view of the proteomic landscape during differentiation of ReNcell VM cells, an immortalized human neural progenitor line.

Sci Data 2019 02 19;6:190016. Epub 2019 Feb 19.

Laboratory of Systems Pharmacology, Program in Therapeutic Science, Harvard Medical School, Boston, MA 02115, USA.

The immortalized human ReNcell VM cell line represents a reproducible and easy-to-propagate cell culture system for studying the differentiation of neural progenitors. To better characterize the starting line and its subsequent differentiation, we assessed protein and phospho-protein levels and cell morphology over a 15-day period during which ReNcell progenitors differentiated into neurons, astrocytes and oligodendrocytes. Five of the resulting datasets measured protein levels or states of phosphorylation based on tandem-mass-tag (TMT) mass spectrometry and four datasets characterized cellular phenotypes using high-content microscopy. Proteomic analysis revealed reproducible changes in pathways responsible for cytoskeletal rearrangement, cell phase transitions, neuronal migration, glial differentiation, neurotrophic signalling and extracellular matrix regulation. Proteomic and imaging data revealed accelerated differentiation in cells treated with the poly-selective CDK and GSK3 inhibitor kenpaullone or the HMG-CoA reductase inhibitor mevastatin, both of which have previously been reported to promote neural differentiation. These data provide in-depth information on the ReNcell progenitor state and on neural differentiation in the presence and absence of drugs, setting the stage for functional studies.
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http://dx.doi.org/10.1038/sdata.2019.16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6380223PMC
February 2019

The NAD Salvage Pathway Supports PHGDH-Driven Serine Biosynthesis.

Cell Rep 2018 08;24(9):2381-2391.e5

Department of Pathology, Dalhousie University, Halifax, NS, Canada; Department of Microbiology and Immunology, Dalhousie University, Halifax, NS, Canada; Centre for Innovative and Collaborative Health Services Research, IWK Health Centre, Halifax, NS, Canada; Department of Biology, Dalhousie University, Halifax, NS, Canada. Electronic address:

NAD is a key metabolic redox cofactor that is regenerated from nicotinamide through the NAD salvage pathway. Here, we find that inhibiting the NAD salvage pathway depletes serine biosynthesis from glucose by impeding the NAD-dependent protein, 3-phosphoglycerate dehydrogenase (PHGDH). Importantly, we find that PHGDH breast cancer cell lines are exquisitely sensitive to inhibition of the NAD salvage pathway. Further, we find that PHGDH protein levels and those of the rate-limiting enzyme of NAD salvage, NAMPT, correlate in ER-negative, basal-like breast cancers. Although NAD salvage pathway inhibitors are actively being pursued in cancer treatment, their efficacy has been poor, and our findings suggest that they may be effective for PHGDH-dependent cancers.
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http://dx.doi.org/10.1016/j.celrep.2018.07.086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6402325PMC
August 2018

Role of Selenof as a Gatekeeper of Secreted Disulfide-Rich Glycoproteins.

Cell Rep 2018 05;23(5):1387-1398

Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Selenof (15-kDa selenoprotein; Sep15) is an endoplasmic reticulum (ER)-resident thioredoxin-like oxidoreductase that occurs in a complex with UDP-glucose:glycoprotein glucosyltransferase. We found that Selenof deficiency in mice leads to elevated levels of non-functional circulating plasma immunoglobulins and increased secretion of IgM during in vitro splenic B cell differentiation. However, Selenof knockout animals show neither enhanced bacterial killing capacity nor antigen-induced systemic IgM activity, suggesting that excess immunoglobulins are not functional. In addition, ER-to-Golgi transport of a target glycoprotein was delayed in Selenof knockout embryonic fibroblasts, and proteomic analyses revealed that Selenof deficiency is primarily associated with antigen presentation and ER-to-Golgi transport. Together, the data suggest that Selenof functions as a gatekeeper of immunoglobulins and, likely, other client proteins that exit the ER, thereby supporting redox quality control of these proteins.
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http://dx.doi.org/10.1016/j.celrep.2018.04.009DOI Listing
May 2018

MS3-IDQ: Utilizing MS3 Spectra beyond Quantification Yields Increased Coverage of the Phosphoproteome in Isobaric Tag Experiments.

J Proteome Res 2018 04 26;17(4):1741-1747. Epub 2018 Feb 26.

Laboratory of Systems Pharmacology , Harvard Medical School , Boston , Massachusetts 02115 , United States.

Protein phosphorylation is critically important for many cellular processes, including progression through the cell cycle, cellular metabolism, and differentiation. Isobaric labeling, for example, tandem mass tags (TMT), in phosphoproteomics workflows enables both relative and absolute quantitation of these phosphorylation events. Traditional TMT workflows identify peptides using fragment ions at the MS2 level and quantify reporter ions at the MS3 level. However, in addition to the TMT reporter ions, MS3 spectra also include fragment ions that can be used to identify peptides. Here we describe using MS3 spectra for both phosphopeptide identification and quantification, a process that we term MS3-IDQ. To maximize quantified phosphopeptides, we optimize several instrument parameters, including the modality of mass analyzer (i.e., ion trap or Orbitrap), MS2 automatic gain control (AGC), and MS3 normalized collision energy (NCE), to achieve the best balance of identified and quantified peptides. Our optimized MS3-IDQ method included the following parameters for the MS3 scan: NCE = 37.5 and AGC target = 1.5 × 10, and scan range = 100-2000. Data from the MS3 scan were complementary to those of the MS2 scan, and the combination of these scans can increase phosphoproteome coverage by >50%, thereby yielding a greater number of quantified and accurately localized phosphopeptides.
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http://dx.doi.org/10.1021/acs.jproteome.8b00006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5984591PMC
April 2018

Aminoglycoside-driven biosynthesis of selenium-deficient Selenoprotein P.

Sci Rep 2017 06 29;7(1):4391. Epub 2017 Jun 29.

Institut für Experimentelle Endokrinologie, Charité-Universitätsmedizin Berlin, Berlin, D-13353, Germany.

Selenoprotein biosynthesis relies on the co-translational insertion of selenocysteine in response to UGA codons. Aminoglycoside antibiotics interfere with ribosomal function and may cause codon misreading. We hypothesized that biosynthesis of the selenium (Se) transporter selenoprotein P (SELENOP) is particularly sensitive to antibiotics due to its ten in frame UGA codons. As liver regulates Se metabolism, we tested the aminoglycosides G418 and gentamicin in hepatoma cell lines (HepG2, Hep3B and Hepa1-6) and in experimental mice. In vitro, SELENOP levels increased strongly in response to G418, whereas expression of the glutathione peroxidases GPX1 and GPX2 was marginally affected. Se content of G418-induced SELENOP was dependent on Se availability, and was completely suppressed by G418 under Se-poor conditions. Selenocysteine residues were replaced mainly by cysteine, tryptophan and arginine in a codon-specific manner. Interestingly, in young healthy mice, antibiotic treatment failed to affect Selenop biosynthesis to a detectable degree. These findings suggest that the interfering activity of aminoglycosides on selenoprotein biosynthesis can be severe, but depend on the Se status, and other parameters likely including age and general health. Focused analyses with aminoglycoside-treated patients are needed next to evaluate a possible interference of selenoprotein biosynthesis by the antibiotics and elucidate potential side effects.
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http://dx.doi.org/10.1038/s41598-017-04586-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5491492PMC
June 2017

A Strategy to Combine Sample Multiplexing with Targeted Proteomics Assays for High-Throughput Protein Signature Characterization.

Mol Cell 2017 Jan 5;65(2):361-370. Epub 2017 Jan 5.

Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Targeted mass spectrometry assays for protein quantitation monitor peptide surrogates, which are easily multiplexed to target many peptides in a single assay. However, these assays have generally not taken advantage of sample multiplexing, which allows up to ten analyses to occur in parallel. We present a two-dimensional multiplexing workflow that utilizes synthetic peptides for each protein to prompt the simultaneous quantification of >100 peptides from up to ten mixed sample conditions. We demonstrate that targeted analysis of unfractionated lysates (2 hr) accurately reproduces the quantification of fractionated lysates (72 hr analysis) while obviating the need for peptide detection prior to quantification. We targeted 131 peptides corresponding to 69 proteins across all 60 National Cancer Institute cell lines in biological triplicate, analyzing 180 samples in only 48 hr (the equivalent of 16 min/sample). These data further elucidated a correlation between the expression of key proteins and their cellular response to drug treatment.
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http://dx.doi.org/10.1016/j.molcel.2016.12.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5250569PMC
January 2017

Neutral Loss Is a Very Common Occurrence in Phosphotyrosine-Containing Peptides Labeled with Isobaric Tags.

J Proteome Res 2017 02 30;16(2):1069-1076. Epub 2016 Dec 30.

Department of Cell Biology, Harvard Medical School , Boston, Massachusetts 02115, United States.

While developing a multiplexed phosphotyrosine peptide quantification assay, an unexpected observation was made: significant neutral loss from phosphotyrosine (pY) containing peptides. Using a 2000-member peptide library, we sought to systematically investigate this observation by comparing unlabeled peptides with the two highest-plex isobaric tags (iTRAQ8 and TMT10) across CID, HCD, and ETD fragmentation using high resolution high mass accuracy Orbitrap instrumentation. We found pY peptide neutral loss behavior was consistent with reduced proton mobility, and does not occur during ETD. The site of protonation at the peptide N-terminus changes from a primary to a tertiary amine as a result of TMT labeling which would increase the gas phase basicity and reduce proton mobility at this site. This change in fragmentation behavior has implications during instrument method development and interpretation of MS/MS spectra, and therefore ensuing follow-up studies. We show how sites not localized to tyrosine by search and site localization algorithms can be confidently reassigned to tyrosine using neutral loss and phosphotyrosine immonium ions. We believe these findings will be of general interest to those studying pY signal transduction using isobaric tags.
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http://dx.doi.org/10.1021/acs.jproteome.6b00487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5479408PMC
February 2017

Quantitative mass spectrometry-based multiplexing compares the abundance of 5000 S. cerevisiae proteins across 10 carbon sources.

J Proteomics 2016 10 16;148:85-93. Epub 2016 Jul 16.

Department of Cell Biology, Harvard Medical School, Boston, MA 02115, United States. Electronic address:

Unlabelled: The budding yeast Saccharomyces cerevisiae is a model system for investigating biological processes. Cellular events are known to be dysregulated due to shifts in carbon sources. However, the comprehensive proteomic alterations thereof have not been fully investigated. Here we examined proteomic alterations in S. cerevisiae due to the adaptation of yeast from glucose to nine different carbon sources - maltose, trehalose, fructose, sucrose, glycerol, acetate, pyruvate, lactic acid, and oleate. Isobaric tag-based mass spectrometry techniques are at the forefront of global proteomic investigations. As such, we used a TMT10-plex strategy to study multiple growth conditions in a single experiment. The SPS-MS3 method on an Orbitrap Fusion Lumos mass spectrometer enabled the quantification of over 5000 yeast proteins across ten carbon sources at a 1% protein-level FDR. On average, the proteomes of yeast cultured in fructose and sucrose deviated the least from those cultured in glucose. As expected, gene ontology classification revealed the major alteration in protein abundances occurred in metabolic pathways and mitochondrial proteins. Our protocol lays the groundwork for further investigation of carbon source-induced protein alterations. Additionally, these data offer a hypothesis-generating resource for future studies aiming to investigate both characterized and uncharacterized genes.

Biological Significance: We investigate the proteomic alterations in S. cerevisiae resulting from adaptation of yeast from glucose to nine different carbon sources - maltose, trehalose, fructose, sucrose, glycerol, acetate, pyruvate, lactic acid, and oleate. SPS-MS3 TMT10plex analysis is used for quantitative proteomic analysis. We showcase a technique that allows the quantification of over 5000 yeast proteins, the highest number to date in S. cerevisiae, across 10 growth conditions in a single experiment. As expected, gene ontology classification of proteins with the major alterations in abundances occurred in metabolic pathways and mitochondrial proteins, reflecting the degree of metabolic stress when cellular machinery shifts from growth on glucose to an alternative carbon source. Our protocol lays the groundwork for further investigation of carbon source-induced protein alterations. Improving depth of coverage - measuring abundance changes of over 5000 proteins - increases our understanding of difficult-to-study genes in the model system S. cerevisiae and by homology human cell biology. We submit this highly comprehensive dataset as a hypothesis generating resource for targeted studies on uncharacterized genes.
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http://dx.doi.org/10.1016/j.jprot.2016.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5035620PMC
October 2016

Regulation of Selenocysteine Content of Human Selenoprotein P by Dietary Selenium and Insertion of Cysteine in Place of Selenocysteine.

PLoS One 2015 9;10(10):e0140353. Epub 2015 Oct 9.

Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, 02115, United States of America.

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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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0140353PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4599804PMC
June 2016

Comprehensive Temporal Protein Dynamics during the Diauxic Shift in Saccharomyces cerevisiae.

Mol Cell Proteomics 2015 Sep 15;14(9):2454-65. Epub 2015 Jun 15.

From the ‡Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, 02115

Yeast (Saccharomyces cerevisiae) has served as a key model system in biology and as a benchmark for "omics" technology. Although near-complete proteomes of log phase yeast have been measured, protein abundance in yeast is dynamic, particularly during the transition from log to stationary phase. Defining the dynamics of proteomic changes during this transition, termed the diauxic shift, is important to understand the basic biology of proliferative versus quiescent cells. Here, we perform temporal quantitative proteomics to fully capture protein induction and repression during the diauxic shift. Accurate and sensitive quantitation at a high temporal resolution and depth of proteome coverage was achieved using TMT10 reagents and LC-MS3 analysis on an Orbitrap Fusion tribrid mass spectrometer deploying synchronous precursor selection. Triplicate experiments were analyzed using the time-course R package and a simple template matching strategy was used to reveal groups of proteins with similar temporal patterns of protein induction and repression. Within these groups are functionally distinct types of proteins such as those of glyoxylate metabolism and many proteins of unknown function not previously associated with the diauxic shift (e.g. YNR034W-A and FMP16). We also perform a dual time-course experiment to determine Hap2-dependent proteins during the diauxic shift. These data serve as an important basic model for fermentative versus respiratory growth of yeast and other eukaryotes and are a benchmark for temporal quantitative proteomics.
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http://dx.doi.org/10.1074/mcp.M114.045849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4563728PMC
September 2015

Quantitative Proteomic Atlas of Ubiquitination and Acetylation in the DNA Damage Response.

Mol Cell 2015 Sep 4;59(5):867-81. Epub 2015 Jun 4.

Department of Genetics, Harvard Medical School; Division of Genetics, Brigham and Women's Hospital; Howard Hughes Medical Institute, Boston, MA 02115, USA. Electronic address:

Execution of the DNA damage response (DDR) relies upon a dynamic array of protein modifications. Using quantitative proteomics, we have globally profiled ubiquitination, acetylation, and phosphorylation in response to UV and ionizing radiation. To improve acetylation site profiling, we developed the strategy FACET-IP. Our datasets of 33,500 ubiquitination and 16,740 acetylation sites provide valuable insight into DDR remodeling of the proteome. We find that K6- and K33-linked polyubiquitination undergo bulk increases in response to DNA damage, raising the possibility that these linkages are largely dedicated to DDR function. We also show that Cullin-RING ligases mediate 10% of DNA damage-induced ubiquitination events and that EXO1 is an SCF-Cyclin F substrate in the response to UV radiation. Our extensive datasets uncover additional regulated sites on known DDR players such as PCNA and identify previously unknown DDR targets such as CENPs, underscoring the broad impact of the DDR on cellular physiology.
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http://dx.doi.org/10.1016/j.molcel.2015.05.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4560960PMC
September 2015

Mechanism-based proteomic screening identifies targets of thioredoxin-like proteins.

J Biol Chem 2015 Feb 5;290(9):5685-95. Epub 2015 Jan 5.

From the Division of Genetics, Department of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115,

Thioredoxin (Trx)-fold proteins are protagonists of numerous cellular pathways that are subject to thiol-based redox control. The best characterized regulator of thiols in proteins is Trx1 itself, which together with thioredoxin reductase 1 (TR1) and peroxiredoxins (Prxs) comprises a key redox regulatory system in mammalian cells. However, there are numerous other Trx-like proteins, whose functions and redox interactors are unknown. It is also unclear if the principles of Trx1-based redox control apply to these proteins. Here, we employed a proteomic strategy to four Trx-like proteins containing CXXC motifs, namely Trx1, Rdx12, Trx-like protein 1 (Txnl1) and nucleoredoxin 1 (Nrx1), whose cellular targets were trapped in vivo using mutant Trx-like proteins, under conditions of low endogenous expression of these proteins. Prxs were detected as key redox targets of Trx1, but this approach also supported the detection of TR1, which is the Trx1 reductant, as well as mitochondrial intermembrane proteins AIF and Mia40. In addition, glutathione peroxidase 4 was found to be a Rdx12 redox target. In contrast, no redox targets of Txnl1 and Nrx1 could be detected, suggesting that their CXXC motifs do not engage in mixed disulfides with cellular proteins. For some Trx-like proteins, the method allowed distinguishing redox and non-redox interactions. Parallel, comparative analyses of multiple thiol oxidoreductases revealed differences in the functions of their CXXC motifs, providing important insights into thiol-based redox control of cellular processes.
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http://dx.doi.org/10.1074/jbc.M114.597245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4342480PMC
February 2015

Evaluating multiplexed quantitative phosphopeptide analysis on a hybrid quadrupole mass filter/linear ion trap/orbitrap mass spectrometer.

Anal Chem 2015 Jan 6;87(2):1241-9. Epub 2015 Jan 6.

Harvard Medical School , Department of Cell Biology, Boston, Massachusetts 02115, United States.

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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http://dx.doi.org/10.1021/ac503934fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4303329PMC
January 2015

Effects of MEK inhibitors GSK1120212 and PD0325901 in vivo using 10-plex quantitative proteomics and phosphoproteomics.

Proteomics 2015 Jan 18;15(2-3):462-73. Epub 2014 Oct 18.

Department of Cell Biology, Harvard Medical School, Boston, MA, USA.

Multiplexed isobaric tag based quantitative proteomics and phosphoproteomics strategies can comprehensively analyze drug treatments effects on biological systems. Given the role of mitogen-activated protein/extracellular signal-regulated kinase (MEK) signaling in cancer and mitogen-activated protein kinase (MAPK)-dependent diseases, we sought to determine if this pathway could be inhibited safely by examining the downstream molecular consequences. We used a series of tandem mass tag 10-plex experiments to analyze the effect of two MEK inhibitors (GSK1120212 and PD0325901) on three tissues (kidney, liver, and pancreas) from nine mice. We quantified ∼ 6000 proteins in each tissue, but significant protein-level alterations were minimal with inhibitor treatment. Of particular interest was kidney tissue, as edema is an adverse effect of these inhibitors. From kidney tissue, we enriched phosphopeptides using titanium dioxide (TiO2 ) and quantified 10 562 phosphorylation events. Further analysis by phosphotyrosine peptide immunoprecipitation quantified an additional 592 phosphorylation events. Phosphorylation motif analysis revealed that the inhibitors decreased phosphorylation levels of proline-x-serine-proline (PxSP) and serine-proline (SP) sites, consistent with extracellular-signal-regulated kinase (ERK) inhibition. The MEK inhibitors had the greatest decrease on the phosphorylation of two proteins, Barttin and Slc12a3, which have roles in ion transport and fluid balance. Further studies will provide insight into the effect of these MEK inhibitors with respect to edema and other adverse events in mouse models and human patients.
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http://dx.doi.org/10.1002/pmic.201400154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4515953PMC
January 2015

Selenoprotein S is involved in maintenance and transport of multiprotein complexes.

Biochem J 2014 Sep;462(3):555-65

*Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, U.S.A.

SelS (Selenoprotein S) is a selenocysteine-containing protein with roles in ER (endoplasmic reticulum) function and inflammation. It has been implicated in ERAD (ER-associated protein degradation), and clinical studies revealed an association of its promoter polymorphism with cytokine levels and human diseases. However, the pathways and interacting proteins that could shed light on pathogenesis of SelS-associated diseases have not been studied systematically. We performed a large-scale affinity isolation of human SelS and its mutant forms and analysed the proteins that interact with them. All previously known SelS targets and nearly two hundred additional proteins were identified that were remarkably enriched for various multiprotein complexes. Subsequent chemical cross-linking experiments identified the specific interacting sites in SelS and its several targets. Most of these interactions involved coiled-coil domains. The data suggest that SelS participates in intracellular membrane transport and maintenance of protein complexes by anchoring them to the ER membrane.
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http://dx.doi.org/10.1042/BJ20140076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5578454PMC
September 2014

Structurally distinct Ca(2+) signaling domains of sperm flagella orchestrate tyrosine phosphorylation and motility.

Cell 2014 May;157(4):808-22

Howard Hughes Medical Institute, Department of Cardiology, Boston Children's Hospital, 320 Longwood Avenue, Boston, MA 02115, USA; Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA. Electronic address:

Spermatozoa must leave one organism, navigate long distances, and deliver their paternal DNA into a mature egg. For successful navigation and delivery, a sperm-specific calcium channel is activated in the mammalian flagellum. The genes encoding this channel (CatSpers) appear first in ancient uniflagellates, suggesting that sperm use adaptive strategies developed long ago for single-cell navigation. Here, using genetics, super-resolution fluorescence microscopy, and phosphoproteomics, we investigate the CatSper-dependent mechanisms underlying this flagellar switch. We find that the CatSper channel is required for four linear calcium domains that organize signaling proteins along the flagella. This unique structure focuses tyrosine phosphorylation in time and space as sperm acquire the capacity to fertilize. In heterogeneous sperm populations, we find unique molecular phenotypes, but only sperm with intact CatSper domains that organize time-dependent and spatially specific protein tyrosine phosphorylation successfully migrate. These findings illuminate flagellar adaptation, signal transduction cascade organization, and fertility.
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http://dx.doi.org/10.1016/j.cell.2014.02.056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4032590PMC
May 2014

Combining amine metabolomics and quantitative proteomics of cancer cells using derivatization with isobaric tags.

Anal Chem 2014 Apr 20;86(7):3585-93. Epub 2014 Mar 20.

Department of Cell Biology, Harvard Medical School , Boston, Massachusetts 02115, United States.

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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http://dx.doi.org/10.1021/ac500153aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3983006PMC
April 2014

Increasing throughput in targeted proteomics assays: 54-plex quantitation in a single mass spectrometry run.

Anal Chem 2013 Jun 23;85(11):5340-6. Epub 2013 May 23.

Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

Targeted proteomics assays such as those measuring end points in activity assays are sensitive and specific but often lack in throughput. In an effort to significantly increase throughput, a comparison was made between the traditional approach which utilizes an internal standard and the multiplexing approach which relies on isobaric tagging. A kinase activity assay was used for proof of concept, and experiments included three biological replicates for every condition. Results from the two approaches were highly similar with the multiplexing showing greater throughput. Two novel 6-plex isobaric tags were added for a total of three 6-plex experiments (18-plex) in a single run. Next, three mass variants of the target peptide were labeled with the three isobaric tags giving nine 6-plex reactions for 54-plex quantitation in a single run. Since the multiplexing approach allows all samples to be combined prior to purification and acquisition, the 54-plex approach resulted in a significant reduction in purification resources (time, reagents, etc.) and a ~50-fold improvement in acquisition throughput. We demonstrate the 54-plex assay in several ways including measuring inhibition of PKA activity in MCF7 cell lysates for a panel of nine compounds.
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http://dx.doi.org/10.1021/ac400845eDOI Listing
June 2013

High error rates in selenocysteine insertion in mammalian cells treated with the antibiotic doxycycline, chloramphenicol, or geneticin.

J Biol Chem 2013 May 15;288(21):14709-15. Epub 2013 Apr 15.

Molecular Biology of Selenium Section, Laboratory of Cancer Prevention, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.

Antibiotics target bacteria by interfering with essential processes such as translation, but their effects on translation in mammalian cells are less well characterized. We found that doxycycline, chloramphenicol, and Geneticin (G418) interfered with insertion of selenocysteine (Sec), which is encoded by the stop codon, UGA, into selenoproteins in murine EMT6 cells. Treatment of EMT6 cells with these antibiotics reduced enzymatic activities and Sec insertion into thioredoxin reductase 1 (TR1) and glutathione peroxidase 1 (GPx1). However, these proteins were differentially affected due to varying errors in Sec insertion at UGA. In the presence of doxycycline, chloramphenicol, or G418, the Sec-containing form of TR1 decreased, whereas the arginine-containing and truncated forms of this protein increased. We also detected antibiotic-specific misinsertion of cysteine and tryptophan. Furthermore, misinsertion of arginine in place of Sec was commonly observed in GPx1 and glutathione peroxidase 4. TR1 was the most affected and GPx1 was the least affected by these translation errors. These observations were consistent with the differential use of two Sec tRNA isoforms and their distinct roles in supporting accuracy of Sec insertion into selenoproteins. The data reveal widespread errors in inserting Sec into proteins and in dysregulation of selenoprotein expression and function upon antibiotic treatment.
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http://dx.doi.org/10.1074/jbc.M112.446666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3663496PMC
May 2013

Selenoprotein K binds multiprotein complexes and is involved in the regulation of endoplasmic reticulum homeostasis.

J Biol Chem 2011 Dec 20;286(50):42937-48. Epub 2011 Oct 20.

Division of Genetics, Department of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

Selenoprotein K (SelK) is an 11-kDa endoplasmic reticulum (ER) protein of unknown function. Herein, we defined a new eukaryotic protein family that includes SelK, selenoprotein S (SelS), and distantly related proteins. Comparative genomics analyses indicate that this family is the most widespread eukaryotic selenoprotein family. A biochemical search for proteins that interact with SelK revealed ER-associated degradation (ERAD) components (p97 ATPase, Derlins, and SelS). In this complex, SelK showed higher affinity for Derlin-1, whereas SelS had higher affinity for Derlin-2, suggesting that these selenoproteins could determine the nature of the substrate translocated through the Derlin channel. SelK co-precipitated with soluble glycosylated ERAD substrates and was involved in their degradation. Its gene contained a functional ER stress response element, and its expression was up-regulated by conditions that induce the accumulation of misfolded proteins in the ER. Components of the oligosaccharyltransferase complex (ribophorins, OST48, and STT3A) and an ER chaperone, calnexin, were found to bind SelK. A glycosylated form of SelK was also detected, reflecting its association with the oligosaccharyltransferase complex. These data suggest that SelK is involved in the Derlin-dependent ERAD of glycosylated misfolded proteins and that the function defined by the prototypic SelK is the widespread function of selenium in eukaryotes.
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http://dx.doi.org/10.1074/jbc.M111.310920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3234841PMC
December 2011

Comparative analysis of Erk phosphorylation suggests a mixed strategy for measuring phospho-form distributions.

Mol Syst Biol 2011 Apr;7:482

Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.

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.
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http://dx.doi.org/10.1038/msb.2011.15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3097084PMC
April 2011

Targeted insertion of cysteine by decoding UGA codons with mammalian selenocysteine machinery.

Proc Natl Acad Sci U S A 2010 Dec 29;107(50):21430-4. Epub 2010 Nov 29.

Molecular Biology of Selenium Section, Laboratory of Cancer Prevention, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Cysteine (Cys) is inserted into proteins in response to UGC and UGU codons. Herein, we show that supplementation of mammalian cells with thiophosphate led to targeted insertion of Cys at the UGA codon of thioredoxin reductase 1 (TR1). This Cys was synthesized by selenocysteine (Sec) synthase on tRNA([Ser]Sec) and its insertion was dependent on the Sec insertion sequence element in the 3'UTR of TR1 mRNA. The substrate for this reaction, thiophosphate, was synthesized by selenophosphate synthetase 2 from ATP and sulfide and reacted with phosphoseryl-tRNA([Ser]Sec) to generate Cys-tRNA([Ser]Sec). Cys was inserted in vivo at UGA codons in natural mammalian TRs, and this process was regulated by dietary selenium and availability of thiophosphate. Cys occurred at 10% of the Sec levels in liver TR1 of mice maintained on a diet with normal amounts of selenium and at 50% in liver TR1 of mice maintained on a selenium deficient diet. These data reveal a novel Sec machinery-based mechanism for biosynthesis and insertion of Cys into protein at UGA codons and suggest new biological functions for thiophosphate and sulfide in mammals.
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http://dx.doi.org/10.1073/pnas.1009947107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3003055PMC
December 2010

Characterization of Clostridium species utilizing liquid chromatography/mass spectrometry of intact proteins.

J Microbiol Methods 2009 May 6;77(2):152-8. Epub 2009 Feb 6.

Commonwealth of Virginia, Division of Consolidated Laboratory Services, 600 N. 5th Street, Richmond, VA 23219, USA.

A method for biomarker candidate discovery and strain level pathogen characterization using liquid chromatography/mass spectrometry (LC/MS) with electrospray ionization is described. This method was applied to two pathogenic Clostridium species: C. difficile and C. perfringens. Seven marker proteins per species (fourteen total) were successfully implemented to speciate unknowns during a blind study and could enhance serological and genetic approaches by serving as new targets for detection. Two sets of C. perfringens isolates that were 100% similar by pulsed-field gel electrophoresis (PFGE) were distinguished using LC/MS, demonstrating the high specificity of this approach. The use of LC/MS is less labor intensive than PFGE, affords greater specificity than real-time PCR, and requires no primers or antibodies.
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http://dx.doi.org/10.1016/j.mimet.2009.01.013DOI Listing
May 2009