Upload
emmats
View
2.097
Download
0
Embed Size (px)
Citation preview
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 [email protected] [490.00-2000.00]
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
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 [email protected] [490.00-2000.00]
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
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)
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
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
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
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
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
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
0.02
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
-0.02
-0.01
0.00
0.01
0.02
Axis 1
Axis
2
High1
High2High3
High4
HighMS1
HighMS2HighMS3
HighMS4
Low1
Low2
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