Using  Proteomics  to  Understand  Crassostrea   gigas’s  Response  to    Multiple  Stressors	     Emma Timmins-Schiffman ...
Ocean  Acidification	•  Current and projected changes in pCO2 are   unprecedented                                          ...
Ocean  Acidification  and          Bivalves	          pCO2                     Energy/resource use                 Growth &...
Characterize how ocean acidification  affects the oyster’sresponse to a second        stressor
427 µatm638 µatm810 µatm991 µatm1182 µatm2848 µatm
Experimental  Design	                                pCO2 (µatm)                   400   600    800     1000   1200   2800...
Experimental  Design	                              pCO2 (µatm)                 400   600    800     1000     1200   2800  ...
Experimental  Design	 •  Mechanical stress as a proxy for environmental    stress •  MS promotes a catecholmine stress res...
Methods:  Proteomics	                   2012_0301_PARP1_plusBOV_10 #7891 RT: 69.73 AV: 1 NL: 3.19E1                       ...
Results:  Proteomics	          S	                •  1,241 proteins were                   identified                •  Cov...
Results:  Proteomics	                 Oxidation-                 reduction is the                 most significantly      ...
pCO2	         0.010                                                        Low1                                           ...
Mechanical  Stress	                                   LowMS2         0.02         0.01                                    ...
0.03                           Mechanical  Stress	                                                                        ...
Mechanical  Stress	                 Venny,  Oliveros  2007
Mechanical  Stress	                 Venny,  Oliveros  2007
pCO2  and  Mechanical                 Stress	•  pH Homeostasis  •    V-type proton ATPase subunit C1 (CGI_10004936)•  Immu...
Summary	Proteomics can provide insight into the physiologicalresponse to ocean acidification.
Summary	Exposure to multiple stressors can impact anorganism’s ability to mount a successful response toeither stressor.
Implications	We should continue to consider multiple stressors when    assessing responses to environmental change.       ...
Acknowledgements	•  Taylor Shellfish: Joth Davis, Jason Ragan, Dustin   Johnson•  Friday Harbor Labs: Emily Carrington, Ke...
E timmins schiffman_nsa2013
E timmins schiffman_nsa2013
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E timmins schiffman_nsa2013

  1. 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. 2. Ocean  Acidification •  Current and projected changes in pCO2 are unprecedented Zeebe  2011
  3. 3. Ocean  Acidification  and   Bivalves pCO2 Energy/resource use Growth & calcification •  Acidosis and shell dissolution •  CO32- availability
  4. 4. Characterize how ocean acidification affects the oyster’sresponse to a second stressor
  5. 5. 427 µatm638 µatm810 µatm991 µatm1182 µatm2848 µatm
  6. 6. Experimental  Design pCO2 (µatm) 400 600 800 1000 1200 2800t0: shell weight 1 month exposureNo additional Mechanicalstress 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. 7. Experimental  Design pCO2 (µatm) 400 600 800 1000 1200 2800 1 month exposureNo additional Mechanicalstress stress •  Shell weight •  Gill tissue: •  Transcriptomics •  Proteomics •  Whole body: fatty acids
  8. 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. 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. 10. Results:  Proteomics S •  1,241 proteins were identified •  Coverage of entire C. gigas proteome: 4.5% Venny,  Oliveros  2007
  11. 11. Results:  Proteomics Oxidation- reduction is the most significantly enriched process !
  12. 12. pCO2 0.010 Low1 Low3 Low2 High1 0.005 pCO2 has a significant effectAxis 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
  13. 13. Mechanical  Stress LowMS2 0.02 0.01 LowMS4 LowMS3Axis 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
  14. 14. 0.03 Mechanical  Stress Proteome 0.02 HighMS1 expression is significantly different at High 0.01 pCO2, but not atAxis 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
  15. 15. Mechanical  Stress Venny,  Oliveros  2007
  16. 16. Mechanical  Stress Venny,  Oliveros  2007
  17. 17. 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)
  18. 18. Summary Proteomics can provide insight into the physiologicalresponse to ocean acidification.
  19. 19. Summary Exposure to multiple stressors can impact anorganism’s ability to mount a successful response toeither stressor.
  20. 20. 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
  21. 21. 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

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