Cliff Gagnier Dr. Dennis Hruby Department of Microbiology Oregon State University - PowerPoint PPT Presentation

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Cliff Gagnier Dr. Dennis Hruby Department of Microbiology Oregon State University

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  1. Pox Proteomics:Identification of the Proteins Associated with theMembranous Fraction of Vaccinia Virus Cliff Gagnier Dr. Dennis Hruby Department of Microbiology Oregon State University

  2. Significance • Vaccinia Virus is used as the vaccine for smallpox • It is estimated that nearly 1/10 of all humans that have ever lived have been infected by smallpox • Bioterrorism • Natural Mutation • Safer Vaccine • Drug Development

  3. Objective • The object of this project is to identify the proteins associated with the membrane and lateral body of the vaccinia virus virion.

  4. membrane lateral body The Vaccinia Virion core

  5. Method: Summary Virus Preparation Mass Spectrometer High Performance Liquid Chromatography (HPLC) Sample Preparation Data Analysis

  6. Add Detergent: Octyl-glucopyranoside (OG) OH O O OH OH OH Method: Sample Preparation *Proteins Not to Scale

  7. Centrifuge Supernatant Pellet Method: Sample Preparation

  8. Method: High Performance Liquid Chromatography (HPLC) Me (Fraction Collector) l = 214 nm l = 254 nm Dual wavelength absorbance detector Chromatogram Pumps

  9. Method: Mass Spectrometer • Measures the molecular mass of molecules - can be as small as H2(2 Da) or as large as a protein(>100,000 Da) • Has many other applications, but the application needed specifically for this project involves determining the amino acid sequence of the peptides in the membranous fraction in order to identify specific proteins • Mass range of the peptides for sequence information is between 500 – 4000 Da • Enzyme digestion – Trypsin (K and R)

  10. H H CH3 CH3 H H H CH2-(CH2)3-NH2 + N C C N C C N C C N C C OH H H O H H O H H O H H O + + + Glycine Alanine Alanine Lysine 218.1 Mass Spectra 346.1 129.0 58.0 147.1 289.1 200.0 0 100 200 300 Method: Sequence Analysis H Molecular Weight of the backbone 57.0 71.0 71.0 128.1

  11. S S E A L G Q S Method: Sequence Analysis SSEALGQS 889.5 719.4 519.3 648.4 432.3 832.5 1017.6 345.2 1104.6

  12. 53 min – 55 min HPLC Chromatogram of the Membranous Fraction of Vaccinia Virus l= 214 nm l= 254 nm

  13. Trypsin Trypsin Digest of Fraction 53 – 55 min

  14. MS Ion Chromatogram for Fraction 53 – 55 min 100 42 min 95 90 85 80 75 70 65 60 55 Relative Abundance 50 45 40 35 30 { 25 20 15 10 5 0 0 5 10 15 20 25 30 35 40 45 50 55 Time (min)

  15. MS1 Spectra at RT = 42 min 1938.8 100 1939.9 95 90 85 80 75 70 970.2 65 60 55 Relative Abundance 50 45 40 35 30 25 1941.8 20 15 1553.8 10 1171.5 1537.9 1251.3 1587.8 1386.8 1874.7 777.8 5 1134.4 982.6 1667.8 859.3 1962.8 495.1 609.2 723.0 0 400 600 800 1000 1200 1400 1600 1800 2000 m/z

  16. MS2 Spectrum for the Largest Peak at RT = 42 min 1219.7 100 95 90 85 80 75 70 65 1332.8 60 55 1218.7 1082.6 50 Relative Abundance 983.6 45 40 35 982.6 30 1331.7 862.8 25 1073.6 859.5 1299.6 868.6 20 1083.6 15 1680.8 957.7 610.5 477.2 1515.6 641.5 755.5 10 723.3 407.4 1445.7 554.4 5 985.6 1158.2 1533.1 929.4 389.9 280.0 1765.6 0 200 400 600 800 1000 1200 1400 1600 m/z

  17. MS2 Spectrum After Mascot Search Againstthe Database for Peptide Homology G4L Score = 57 SEYDILHVDILSFFLK

  18. An Example of Poor Identification A10L Score = 1 TVTNLISETLK

  19. Proteins Found In Fraction 53 – 55 min • A4L • G4L • A27L • E10R Possible Protein Matches made by the Mascot Program Proteins Determined to Exist After Further Analysis of the Data • A4L • G4L • A27L • E10R • D5R • A10L • B18R • C9L

  20. Total Proteins Found In the Membranous Fraction of Vaccinia Virus A4L – Membrane associated core protein G4L – Glutaredoxin A12L – Virion protein G7L – Virion core protein A13L – Structural protein H1L – Tryocine phosphotase A15L – Hypothetical protein H3L – IMV membrane associated protein A27L – Cell fusion protein H5R – Late transcription factor A30L – Vaccinia virus morphogenesis factor J1R – Dimeric virion protein A42R – Profilin-like protein K4L – Hypothetical protein B2R – Hypothetical protein L1R – myristylated membrane protein C16L – Hypothetical protein L3L – Hypothetical protein E10R – Redox protein O2L – Glutaredoxin F8L – Actin disassembly protein F17R – DNA-binding protein

  21. Acknowledgments • Howard Hughes Medical Institute • Dr. Dennis Hruby • The Members of the Hruby Lab • With Special Thanks to Susan Chen and Jennifer Yoder • OSU EHSC Mass Spectrometry Core Facilities • With Special Thanks to Brian Arbogast and Elisabeth Barofsky