1. Pox Proteomics: Identification of the Proteins Associated with the Membranous 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. The Vaccinia Virion membrane lateral body core

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

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

7. Method: Sample Preparation Centrifuge Pellet Supernatant

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

9. Method: Mass Spectrometer Measures the molecular mass of molecules - can be as small as H 2 (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. Method: Sequence Analysis 58.0 129.0 147.1 200.0 289.1 218.1 57.071.0 128.1 Molecular Weight of the backbone 0100200300 Mass Spectra 346.1 H N H C HO CN H C O C CH 3 N H C O C N H C O C CH 2 -(CH 2 ) 3 -NH 2 Glycine Alanine HHH OH Lysine H +++ H H + HH

11. Method: Sequence Analysis SSEALGQS QSGLAES 1017.6 889.5 832.5 1104.6 719.4 648.4 519.3 432.3 345.2 S

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

13. Trypsin Digest of Fraction 53 – 55 min Trypsin

14. MS Ion Chromatogram for Fraction 53 – 55 min 0510152025303540455055 Time (min) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance { 42 min

15. MS1 Spectra at RT = 42 min 400600800100012001400160018002000 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 1938.8 1939.9 970.2 1941.8 1553.8 1171.5 1537.9 1251.3 1587.8 1386.8 1874.7 777.8 1134.4 982.6 1667.8 1962.8859.3 495.1 723.0609.2

16. MS2 Spectrum for the Largest Peak at RT = 42 min 2004006008001000120014001600 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 Relative Abundance 1219.7 1332.8 1218.7 1082.6 983.6 982.6 1331.7 862.8 1073.6 859.5 868.6 1299.6 1083.6 1680.8 957.7 610.5 641.5 477.2 755.5 1515.6 723.3 1445.7 407.4 554.4 985.61158.2 1533.1 929.4 389.9 280.0 1765.6 100

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

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

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

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