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Mass Spectrometry at The University of Louisville HSC

Mass Spectrometry at The University of Louisville HSC. Jon Klein, M.D., Ph.D Director Michael L. Merchant, Ph.D. Technical Director, Proteomics Laboratory Department of Medicine. Background. Biomolecular Mass Spectrometry Core – Established by Bill Pierce ~1997

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Mass Spectrometry at The University of Louisville HSC

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  1. Mass Spectrometry atThe University of Louisville HSC Jon Klein, M.D., Ph.D Director Michael L. Merchant, Ph.D. Technical Director, Proteomics Laboratory Department of Medicine

  2. Background • Biomolecular Mass Spectrometry Core – Established by Bill Pierce ~1997 • Proteomics Core – Established 1998 • Merger of both laboratories into a single HSC Mass Spectrometry Core – 2012

  3. Working definitions • Proteome • the total set of proteins expressed in a biological compartment at a given point in time. Now recognized to include details of quantity and extent of modifications. • Proteomics • The study of the proteome. • The evolution of proteomic research follows three epochs of mass spectrometry development- • First generation (MS) and peptide mass fingerprinting (PMF) • Second generation and use of tandem mass spectrometry (MS/MS or MS2) to obtain amino acid sequence • Third generation and the development of MSnmethods for quantitative and advanced qualitative proteomic data sets

  4. Three Broad Categories of Proteomic Studies • Expression and quantitative proteomics • Define and quantify the protein components: • Functional proteomics. • Define the interactions among proteins: • Protein Interaction Networks (PIN) • Define the mechanisms by which proteins communicate with each other: • Protein Signaling Networks (PSN) Courtesy of K. McLeish (2012)

  5. Steps in Proteomic Analysis • Define the question proteomics will answer • Isolation of protein-containing structure • Protein extraction: sonication, chaotropes, detergents • Protein separation: 2DE, HPLC, LC • Protein digestion: trypsin vs Lys-C • Mass spectrometry analysis • Quantification: label (SILAC, DIGE) or label-free (spectral counting) • Bioinformatics Courtesy of K. McLeish (2012)

  6. Work Station Basic Mass Spectrometer Flight Path

  7. Separation technologies • Three common separation modalities • Electrophoresis • Chromatography • Affinity enrichment (e.g. immunoaffinity) • Why do we need separations • To increase sensitivity for detection of lower abundant proteins.

  8. Most samples have dynamic range of protein expression that exceeds the detector on the mass spectrometer. Consider plasma: Anderson NL, Anderson NG, MCP 1(11) 845-869 2002

  9. Combinations of chromatography and electrophoresis to help observe, identify and quantify low abundant proteins

  10. Whole sample in chicken antibody (IgY) Immunoaffinity removal of abundant proteins to reveal low abundant proteins Inert Bead Fractionated sample out Low abundant proteins first High abundant proteins last Antibodies developed to-

  11. Immunoprecipitation and mass spectrometry analysis of associated-binding proteins Adding protein A makes antibody-protein complex insoluble Add Antibody against Protein of interest Centrifugation of solution Pellets the complex. Removal of supernatant and washing Antibody binds to Protein of interest Search MS/MS data against human protein database w/ or w/o –P modification Acquire tandem-MS data Elute Protein Trypsin digestion Peptide identification Loading on mass spectrometer Courtesy of Madhavi Rane (2012)

  12. Mass spectrometers Best advice on what instrument to use is: Know your goal and collaborate with the MS lab that will select the correct approach for your goal.

  13. Prevailing MS-based Proteomic Method

  14. Effect of improved mass accuracy

  15. High Resolution MS at the HSC • OrbitrapLTQ Velos Elite Mass Spectrometer with ETD capability • Mass Accuracy <3ppm RMS with external calibration, <1ppm RMS using internal calibration • Purchased with VA ShEEP grant and additional funds from the EVPRI, SOM Dean, • Department of Medicine and Division of Nephrology

  16. Costs *Intramural pricing, price for extramural collaborators not listed

  17. HSC Mass Spectrometry Core Team • Michael Merchant • Jian Cai • Danny Wilkey • Ming Li

  18. HSC Mass Spectrometry Core • Goals • Provide innovative state of the art MS • Collaborate • Enhance competitiveness of single and multi-investigator grant applications • Be as self-supporting as possible

  19. Acknowledgements • Bill Pierce • Russ Prough • Department of Veterans Affairs • CEGeMM/ Ron Gregg • Don Miller • Toni Ganzel

  20. Kindly Do Not Forget That Today is “International Talk Like a Pirate Day”

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