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

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

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

The Vaccinia Virion membrane lateral body core

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

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

HPLC Trace of Vaccinia Virus L1R

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

Sequence Analysis Molecular Weight of the amino acids Mass Spectrum 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

Sequence Analysis SSEALGQS QSGLAES S

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

Peak Shift Mass Spectrum 321 GAA GAAC C Mass Spectrum 378 GAA GAAC +IA C +IA

Results: L1R Coverage MGAAASIQTTVNTLSERISSKLEQEANASAQTKCDIE IGNFYIRQNHGCNLTVKNMCSADADAQLDAVLSAA TETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFE NYVKQTCNSSAVVDNKLKIQNVIIDECYGAPGSPTN LEFINTGSSKGNCAIKALMQLTTKATTQIAPRQVAGT GVQFYMIVIGVIILAALFMYYAKRMLFTSTNDKIKLI LANKENVHWTTYMDTFFRTSPMVIATTDMQN

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

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

Results: L1R Coverage MGAAASIQTTVNTLSERISSKLEQEANASAQTKCDIE IGNFYIRQNHGCNLTVKNMCSADADAQLDAVLSAA TETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDF ENYVKQTCNSSAVVDNKLKIQNVIIDECYGAPGSPT NLEFINTGSSKGNCAIKALMQLTTKATTQIAPRQVA GTGVQFYMIVIGVIILAALFMYYAKRMLFTSTNDKIK LILANKENVHWTTYMDTFFRTSPMVIATTDMQN

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

Results: L1R Coverage MGAAASIQTTVNTLSERISSKLEQEANASAQTKCDIE IGNFYIRQNHGCNLTVKNMCSADADAQLDAVLSAA TETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFE NYVKQTCNSSAVVDNKLKIQNVIIDECYGAPGSPTNL EFINTGSSKGNCAIKALMQLTTKATTQIAPRQVAGTG VQFYMIVIGVIILAALFMYYAKRMLFTSTNDKIKLIL ANKENVHWTTYMDTFFRTSPMVIATTDMQN

CDIEIGNFYIR Results: Cysteine Modification Score: 39 IA Unmodified (MW in Da) Modified (MW in Da) b(2) b(3) b(4) b(5)

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

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