1. The Disulfide Bonding Pattern of Vaccinia Virus Protein L1R Cliff Gagnier Dr. Dennis Hruby’s Lab September 1, 2004

2. Significance Vaccinia Virus is used as the vaccine for smallpox Bioterrorism Natural Mutation Safer Vaccine Drug Development

3. The Vaccinia Virion membrane lateral body core

4. L1R Objective To identify the L1R cysteine residues involved in disulfide bonding by mass spectrometry

5. High Performance Liquid Chromatography (HPLC) Chromatogram Pumps Dual wavelength absorbance detector = 214 nm = 254 nm

6. HPLC Trace of Vaccinia Virus L1R

7. NEM - SH - S S S H H A A L1R Experimental Design 1. Add NEM 2. Add Trypsin 3. Add DTT 4. Add Iodoacetamide

8. Sequence Analysis 58.0 129.0 147.1 200.0 289.1 218.1 57.071.0 128.1 Molecular Weight of the amino acids 0100200300 Mass Spectrum 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

9. Sequence Analysis SSEALGQS QSGLAES 1017.6 889.5 832.5 1104.6 719.4 648.4 519.3 432.3 345.2 S

10. Chemical Modifiers n-ethyl-maleimide Iodoacetamide Molecular Weight Addition: 125 Da Molecular Weight Addition: 57 Da Able to search Mascot for cysteines modified with NEM and Iodoacetamide

11. Peak Shift 58.0 129.0 200.0 0100200300 Mass Spectrum 321 GAA GAAC C 58.0 129.0 200.0 0100200300 Mass Spectrum 378 GAA GAAC +IA C +IA

12. Results: L1R Coverage MGAAASIQTTVNTLSERISSKLEQEANASAQTKCDIE IGNFYIRQNHGCNLTVKNMCSADADAQLDAVLSAA TETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFE NYVKQTCNSSAVVDNKLKIQNVIIDECYGAPGSPTN LEFINTGSSKGNCAIKALMQLTTKATTQIAPRQVAGT GVQFYMIVIGVIILAALFMYYAKRMLFTSTNDKIKLI LANKENVHWTTYMDTFFRTSPMVIATTDMQN

13. NEM - SH - S S S L1R Experimental Design 2. Add NEM 1. Add Trypsin

14. H - S S S H H L1R Experimental Design 2. Add Trypsin 1. Add DTT

15. Results: L1R Coverage MGAAASIQTTVNTLSERISSKLEQEANASAQTKCDIE IGNFYIRQNHGCNLTVKNMCSADADAQLDAVLSAA TETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDF ENYVKQTCNSSAVVDNKLKIQNVIIDECYGAPGSPT NLEFINTGSSKGNCAIKALMQLTTKATTQIAPRQVA GTGVQFYMIVIGVIILAALFMYYAKRMLFTSTNDKIK LILANKENVHWTTYMDTFFRTSPMVIATTDMQN

16. H - S S S H H L1R Experimental Design 3. Add Trypsin 1. Add DTT 2. Add Iodoacetamide A A A - S

17. Results: L1R Coverage MGAAASIQTTVNTLSERISSKLEQEANASAQTKCDIE IGNFYIRQNHGCNLTVKNMCSADADAQLDAVLSAA TETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFE NYVKQTCNSSAVVDNKLKIQNVIIDECYGAPGSPTNL EFINTGSSKGNCAIKALMQLTTKATTQIAPRQVAGTG VQFYMIVIGVIILAALFMYYAKRMLFTSTNDKIKLIL ANKENVHWTTYMDTFFRTSPMVIATTDMQN

18. CDIEIGNFYIR Results: Cysteine Modification Score: 39 IA Unmodified (MW in Da) 219332461574 Modified (MW in Da) 276389518631 b(2) b(3) b(4) b(5) 276.0 389.1 518.2 631.3

19. Conclusions & Future Work Inadequate L1R in the sample Double the total vaccinia virus increases with existing protocol 2-D HPLC in conjunction with the Mass Spectrometer If coverage of L1R does exceed 50%, denature with urea

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