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