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

Pox Proteomics:Identification of the Proteins Associated with the

Membranous Fraction of Vaccinia Virus

Cliff Gagnier

Dr. Dennis HrubyDepartment of Microbiology

Oregon State University

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

Objective

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

The Vaccinia Virion

membranelateral body

core

Method: Summary

Virus Preparation

Sample Preparation

High Performance

Liquid Chromatography

(HPLC)

Mass Spectrometer

Data Analysis

Method: Sample Preparation

*Proteins Not to Scale

Add Detergent:

Octyl-glucopyranoside

(OG)O O

OH

OH

OH

OH

Method: Sample Preparation

Centrifuge

Pellet

Supernatant

Method: High Performance Liquid Chromatography (HPLC)

Chromatogram

Me

(Fraction Collector)

Pumps

Dual wavelength absorbance

detector

= 214 nm

= 254 nm

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)

Method: Sequence Analysis

58.0129.0

147.1200.0 289.1

218.1

57.0 71.0 71.0 128.1Molecular Weight of

the backbone

0 100 200 300

Mass Spectra

346.1

H N

H

C

H O

C N

H

C

O

C

CH3

N

H

C

O

C

CH3

N

H

C

O

C

CH2-(CH2)3-NH2

Glycine Alanine Alanine

H H H

OH

Lysine

H + ++

HH

+HH

Method: Sequence Analysis

SSEALGQS

Q SGLAES

1017.6

889.5

832.5

1104.6

719.4

648.4519.3

432.3345.2

S

HPLC Chromatogram of the Membranous Fraction of Vaccinia Virus

072203S Date Acquired: 7/22/03 1:38:36 PM Vial: 1 Inj. #: 1 Channel Desc.:

AU

-0.20

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

2.00

2.20

2.40

2.60

2.80

3.00

3.20

3.40

Minutes

0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00

53 min – 55 min

= 214 nm

= 254 nm

Trypsin Digest of Fraction 53 – 55 min

Trypsin

MS Ion Chromatogram for Fraction 53 – 55 min

0 5 10 15 20 25 30 35 40 45 50 55Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

{

42 min

MS1 Spectra at RT = 42 min

400 600 800 1000 1200 1400 1600 1800 2000m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

1938.8

1939.9

970.2

1941.8

1553.8

1171.5 1537.91251.3 1587.81386.8 1874.7777.8 1134.4982.6 1667.8 1962.8859.3495.1 723.0609.2

MS2 Spectrum for the Largest Peak at RT = 42 min

200 400 600 800 1000 1200 1400 1600

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

Rel

ativ

e A

bund

ance

1219.7

1332.8

1218.71082.6983.6

982.61331.7862.8

1073.6859.5 868.6 1299.6

1083.6 1680.8957.7610.5 641.5477.2755.5

1515.6723.3 1445.7407.4 554.4 985.6 1158.2 1533.1929.4389.9280.0 1765.6

100

MS2 Spectrum After Mascot Search Againstthe Database for Peptide Homology

SEYDILHVDILSFFLK

G4L

Score = 57

An Example of Poor Identification

A10L Score = 1

TVTNLISETLK

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

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

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