Transcript Pox Proteomics: Isolation of membranous fraction

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
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
lateral
body
membrane
core
Method: Summary
Virus
Preparation
Mass
Spectrometer
High
Performance
Liquid
Chromatography
(HPLC)
Sample
Preparation
Data Analysis
Method: Sample Preparation
Add Detergent:
Octylglucopyranoside
(OG)
OH
O
OH
OH
*Proteins Not to Scale
O
OH
Method: Sample Preparation
Centrifuge
Supernatant
Pellet
Method: High Performance Liquid
Chromatography (HPLC)
Me
(Fraction Collector)
l = 214 nm
l = 254 nm
Pumps
Dual
wavelength
absorbance
detector
Chromatogram
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
H
H CH3
H CH3
H
H CH2-(CH2)3-NH2
+
H N C C N C C N C C N C C OH
H H +O H H +O H H +O H H O
Molecular Weight of
the backbone
Glycine
Alanine
Alanine
Lysine
57.0
71.0
71.0
128.1
218.1
Mass
Spectra
346.1
129.0
147.1
58.0
200.0
0
100
200
289.1
300
Method: Sequence Analysis
SSEALGQS
889.5
719.4
519.3
648.4
432.3
832.5
345.2
S
S
E
A
L
G
1017.6
Q
S 1104.6
HPLC Chromatogram of the Membranous
Fraction of Vaccinia Virus
COSINe-Microbiology
Project Name:
Reported by User:
Virion
System
3.40
3.20
3.00
l= 214 nm
2.80
2.60
2.40
2.20
2.00
53 min – 55 min
AU
1.80
1.60
1.40
1.20
1.00
l= 254 nm
0.80
0.60
0.40
0.20
0.00
-0.20
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
Minutes
072203S Date Acquired: 7/22/03 1:38:36 PM Vial: 1 Inj. #: 1 Channel Desc.:
072203S Date Acquired: 7/22/03 1:38:36 PM Vial: 1 Inj. #: 1 Channel Desc.:
100.00
110.00
Trypsin Digest of Fraction 53 – 55 min
Trypsin
MS Ion Chromatogram for Fraction
53 – 55 min
100
42 min
95
90
85
80
75
70
Relative Abundance
65
60
55
50
45
40
35
30
{
25
20
15
10
5
0
0
5
10
15
20
25
30
35
Time (min)
40
45
50
55
MS1 Spectra at RT = 42 min
1938.8
100
1939.9
95
90
85
80
75
70
970.2
Relative Abundance
65
60
55
50
45
40
35
30
25
1941.8
20
15
1553.8
10
5
495.1
777.8
609.2
723.0
859.3
982.6
1171.5
1134.4
1251.3
1386.8
1537.9
1587.8
1874.7
1667.8
1962.8
0
400
600
800
1000
1200
m/z
1400
1600
1800
2000
MS2 Spectrum for the Largest
Peak at RT = 42 min
1219.7
100
95
90
85
80
75
70
Relative Abundance
65
1332.8
60
55
1082.6 1218.7
50
983.6
45
40
35
30
862.8
25
859.5
20
982.6
1331.7
1073.6
1299.6
868.6
1083.6
15
10
5
280.0
0
200
610.5 641.5
477.2
723.3 755.5
407.4
554.4
389.9
400
600
800
957.7
1680.8
1515.6
1445.7
985.6 1158.2
929.4
1000
m/z
1200
1400
1533.1
1600
1765.6
MS2 Spectrum After Mascot Search Against
the Database for Peptide Homology
G4L
Score = 57
SEYDILHVDILSFFLK
An Example of Poor Identification
A10L
Score = 1
TVTNLISETLK
Proteins Found In Fraction 53 – 55 min
Possible Protein Matches made
by the Mascot Program
A4L
G4L
A27L
E10R
D5R
A10L
B18R
C9L
Proteins Determined to Exist After
Further Analysis of the Data
A4L
G4L
A27L
E10R
Total Proteins Found In the Membranous
Fraction of Vaccinia Virus
A4L – Membrane associated core protein
A12L – Virion protein
A13L – Structural protein
A15L – Hypothetical protein
A27L – Cell fusion protein
A30L – Vaccinia virus morphogenesis factor
A42R – Profilin-like protein
B2R – Hypothetical protein
C16L – Hypothetical protein
E10R – Redox protein
F8L – Actin disassembly protein
F17R – DNA-binding protein
G4L – Glutaredoxin
G7L – Virion core protein
H1L – Tryocine phosphotase
H3L – IMV membrane associated protein
H5R – Late transcription factor
J1R – Dimeric virion protein
K4L – Hypothetical protein
L1R – myristylated membrane protein
L3L – Hypothetical protein
O2L – Glutaredoxin
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