1. Using Proteomics to
Understand Crassostrea
gigas’s Response to
Multiple Stressors
Emma Timmins-Schiffman (UW, SAFS)
Brook Nunn (UW, Med. Chem.)
David Goodlett (UW, Med. Chem.)
Carolyn Friedman (UW, SAFS)
Steven Roberts (UW, SAFS)

2. Ocean Acidification
• Current and projected changes in pCO2 are
unprecedented
Zeebe 2011

3. Ocean Acidification and
Bivalves
pCO2
Energy/resource use
Growth & calcification
• Acidosis and shell dissolution
• CO32- availability

4. Characterize how
ocean acidification
affects the oyster’s
response to a second
stressor

5. 427 µatm
638 µatm
810 µatm
991 µatm
1182 µatm
2848 µatm

6. Experimental Design
pCO2 (µatm)
400 600 800 1000 1200 2800
t0: shell weight
1 month exposure
No additional Mechanical
stress stress Heat shock
• 2 sublethal
temperatures:
• Shell weight 42 & 43°C
• Gill tissue: • 1 lethal
• Transcriptomics temperature:
• Proteomics 44°C
• Whole body: fatty acids

7. Experimental Design
pCO2 (µatm)
400 600 800 1000 1200 2800
1 month exposure
No additional Mechanical
stress stress
• Shell weight
• Gill tissue:
• Transcriptomics
• Proteomics
• Whole body: fatty acids

8. Experimental Design
• Mechanical stress as a proxy for environmental
stress
• MS promotes a catecholmine stress response in C.
gigas (Lacoste et al. 2001)
Proteins?

9. Methods: Proteomics
2012_0301_PARP1_plusBOV_10 #7891 RT: 69.73 AV: 1 NL: 3.19E1
T: ITMS + c NSI d Full ms2 1820.34@cid35.00 [490.00-2000.00]
100
1661.2975
• Shotgun sequencing
95
90
85
80
75
using LC-MS/MS Sequenced spectrum
70
65
60
Relative Abundance
55
50
45
40
• Protein fragments
1915.7020
1426.2880
35
30
25
20 1710.3446
15 979.8818
1102.1235
(peptides) are
1255.5396
10 1349.2131
5
0
600 800 1000 1200 1400 1600 1800 2000
m/z
sequenced
Matched to “spectrum”
• Sequences are in database
identified using a 2012_0301_PARP1_plusBOV_10 #5584-5952 RT: 49.46-52.71 AV: 2 NL: 2.87E1
T: Average spectrum MS2 1240.28 (5584-5952)
100 1221.0632
1344.0381
2012_0301_PARP1_plusBOV_10 #5584-5952 RT: 49.46-52.71 AV: 2 NL: 2.87E1
T: Average spectrum MS2 1240.28 (5584-5952)
100 1221.0632
1344.0381
database of proteins
95 95
90 90
85 85
80 80
75 1178.4297 1178.4297
75
70 70
65 65
60 60
Relative Abundance
Relative Abundance
55 1039.4359 1039.4359
55
50 50
45 45
40 40
1424.5980 1424.5980
35 2012_0301_PARP1_plusBOV_10 #7891 RT: 69.73 AV: 1 NL: 3.19E1
35
1490.5931 1490.5931
30
T: ITMS + c NSI 30 Full ms2 1820.34@cid35.00 [490.00-2000.00]
d
25 25
1912.4304 1912.4304 1661.2975
20 100 20
869.9010 1607.7299 1607.7299
869.9010
15 545.8882 823.7305 545.8882
15 823.7305
479.2288 95 479.2288
10 1851.9578 10 1851.9578
5 90 5
0 0
400 600 800 1000 1200 1400 1600 1800 2000
m/z
2012_0301_PARP1_plusBOV_10 #5584-5952 RT: 49.46-52.71 AV: 2 NL: 2.87E1
85 400 600 800 1000 1200 1400 1600 1800 2000
m/z
T: Average spectrum MS2 1240.28 (5584-5952)
1344.0381 80
100 1221.0632
95 75
90
85 70
80
1178.4297
65
75
70 60
Relative Abundance
65
60
55
Relative Abundance
55 1039.4359
50
50
45 45
40
1424.5980
40
35
1490.5931 1915.7020
30
1426.2880
35
25
1912.4304
20
1607.7299
30
869.9010
15 545.8882 823.7305
479.2288 25
1851.9578
10
5
20 1710.3446
0
400 600 800 1000 1200 1400 1600 1800 2000
m/z
15 979.8818
1102.1235
1255.5396
10 1349.2131
5
0
600 800 1000 1200 1400 1600 1800 2000
m/z

10. Results: Proteomics
S
• 1,241 proteins were
identified
• Coverage of entire C.
gigas proteome: 4.5%
Venny, Oliveros 2007

11. Results: Proteomics
Oxidation-
reduction is the
most significantly
enriched process
!

12. pCO2
0.010
Low1
Low3
Low2
High1
0.005
pCO2 has a
significant effect
Axis 2
0.000
on the
proteome
-0.005
Low4
High2
High4
High3
-0.010
-0.015 -0.010 -0.005 0.000 0.005 0.010 0.015
Axis 1

14. Mechanical Stress
LowMS2
0.02
0.01
LowMS4
LowMS3
Axis 2
0.00
Low4
Low3
LowMS1
-0.01
Low2
Low1
-0.02
-0.02 -0.01 0.00 0.01 0.02 0.03
Axis 1

15. 0.03
Mechanical Stress
Proteome
0.02
HighMS1 expression is
significantly
different at High
0.01
pCO2, but not at
Axis 2
HighMS4 HighMS2
Low pCO2
0.00
HighMS3
High1
High4
High2
-0.01
High3
-0.02
-0.02 -0.01 0.00 0.01 0.02 0.03 0.04
Axis 1

16. Mechanical Stress
Venny, Oliveros 2007

17. Mechanical Stress
Venny, Oliveros 2007

19. pCO2 and Mechanical
Stress
• pH Homeostasis
• V-type proton ATPase subunit C1 (CGI_10004936)
• Immune Response
o Cathepsin L (CGI_10009231)
o Heat shock protein 70 12B (CGI_10024293)
o MAP kinase-activated protein kinase 2 (CGI_10003308)

20. Summary
Proteomics can provide insight into the physiological
response to ocean acidification.

21. Summary
Exposure to multiple stressors can impact an
organism’s ability to mount a successful response to
either stressor.

22. Implications
We should continue to consider multiple stressors when
assessing responses to environmental change.
0.02
LowMS2
HighMS1
0.01
High1
High4
HighMS4
LowMS4
HighMS2
0.00
Axis 2
LowMS3 HighMS3
High2
High3 LowMS1
Low4
Low3
-0.01
Low2
Low1
-0.02
-0.02 -0.01 0.00 0.01 0.02 0.03 0.04
Axis 1

23. Acknowledgements
• Taylor Shellfish: Joth Davis, Jason Ragan, Dustin
Johnson
• Friday Harbor Labs: Emily Carrington, Ken Sebens,
Michael O’Donnell, Matt George, Michelle Herko
• UW SAFS: Sam White, Mackenzie Gavery, Caroline
Storer, Samantha Brombacker, Lisa Crosson, Julian
Olden
• UW Proteomics Resource: Priska von Haller, Jimmy
Eng, Eva Jahan
• Ronen Elad, Sam Garson, Chris Parrish
• Funding: NOAA Saltonstall-Kennedy, RocketHub