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)

Ocean  Acidification •  Current and projected changes in pCO2 are

unprecedented

Zeebe  2011

Ocean  Acidification  and  Bivalves

Growth & calcification •  Acidosis and shell dissolution •  CO3

2- availability

Energy/resource use pCO2

Characterize how ocean acidification affects the oyster’s

response to a second stressor

2848 µatm

638 µatm

1182 µatm

427 µatm

810 µatm 991 µatm

Experimental  Design pCO2 (µatm)

400 600 800 1000 1200 2800

1 month exposure

t0: shell weight

No additional stress

Mechanical stress

•  Shell weight •  Gill tissue:

•  Transcriptomics •  Proteomics

•  Whole body: fatty acids

Heat shock •  2 sublethal

temperatures: 42 & 43°C

•  1 lethal temperature: 44°C

Experimental  Design pCO2 (µatm)

400 600 800 1000 1200 2800

1 month exposure

No additional stress

Mechanical stress

•  Shell weight •  Gill tissue:

•  Transcriptomics

•  Proteomics •  Whole body: fatty acids

Experimental  Design •  Mechanical stress as a proxy for environmental

stress •  MS promotes a catecholmine stress response in C.

gigas (Lacoste et al. 2001)

Proteins?

Methods:  Proteomics •  Shotgun sequencing

using LC-MS/MS •  Protein fragments

(peptides) are sequenced

•  Sequences are identified using a database of proteins

2012_0301_PARP1_plusBOV_10 #7891 RT: 69.73 AV: 1 NL: 3.19E1T: ITMS + c NSI d Full ms2 1820.34@cid35.00 [490.00-2000.00]

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1661.2975

1915.70201426.2880

1710.3446

979.8818 1102.1235 1255.53961349.2131

Sequenced spectrum

2012_0301_PARP1_plusBOV_10 #7891 RT: 69.73 AV: 1 NL: 3.19E1T: ITMS + c NSI d Full ms2 1820.34@cid35.00 [490.00-2000.00]

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1661.2975

1915.70201426.2880

1710.3446

979.8818 1102.1235 1255.53961349.2131

2012_0301_PARP1_plusBOV_10 #5584-5952 RT: 49.46-52.71 AV: 2 NL: 2.87E1T: Average spectrum MS2 1240.28 (5584-5952)

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1344.03811221.0632

1178.4297

1039.4359

1424.5980

1490.5931

1912.4304

1607.7299869.9010545.8882 823.7305

479.22881851.9578

2012_0301_PARP1_plusBOV_10 #5584-5952 RT: 49.46-52.71 AV: 2 NL: 2.87E1T: Average spectrum MS2 1240.28 (5584-5952)

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1344.03811221.0632

1178.4297

1039.4359

1424.5980

1490.5931

1912.4304

1607.7299869.9010545.8882 823.7305

479.22881851.9578

2012_0301_PARP1_plusBOV_10 #5584-5952 RT: 49.46-52.71 AV: 2 NL: 2.87E1T: Average spectrum MS2 1240.28 (5584-5952)

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

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bund

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1344.03811221.0632

1178.4297

1039.4359

1424.5980

1490.5931

1912.4304

1607.7299869.9010545.8882 823.7305

479.22881851.9578

Matched to “spectrum” in database

Results:  Proteomics

•  1,241 proteins were identified

•  Coverage of entire C. gigas proteome: 4.5%

S

Venny,  Oliveros  2007

Results:  Proteomics

!

Oxidation-reduction is the most significantly enriched process

-0.015 -0.010 -0.005 0.000 0.005 0.010 0.015

-0.010

-0.005

0.000

0.005

0.010

Axis 1

Axi

s 2

High1

High2High3High4

Low1Low2Low3

Low4

pCO2

pCO2 has a significant effect on the proteome

Mechanical  Stress

-0.02 -0.01 0.00 0.01 0.02 0.03

-0.02

-0.01

0.00

0.01

0.02

Axis 1

Axi

s 2

Low1

Low2

Low3

Low4

LowMS1

LowMS2

LowMS3

LowMS4

Mechanical  Stress

-0.02 -0.01 0.00 0.01 0.02 0.03 0.04

-0.02

-0.01

0.00

0.01

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0.03

Axis 1

Axi

s 2

High1

High2

High3

High4

HighMS1

HighMS2HighMS3

HighMS4

Proteome expression is significantly different at High pCO2, but not at Low pCO2

Mechanical  Stress

Venny,  Oliveros  2007

Mechanical  Stress

Venny,  Oliveros  2007

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)

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

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

Implications We should continue to consider multiple stressors when

assessing responses to environmental change.

-0.02 -0.01 0.00 0.01 0.02 0.03 0.04

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High1

High2High3

High4

HighMS1

HighMS2HighMS3

HighMS4

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Low3Low4

LowMS1

LowMS2

LowMS3

LowMS4

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