0-+/$& 1-#.#2-/#)$&3(+&$&...

!"#$%&'()*+),#-"&*)&!$.+("*/$.&0-+/$&1-#.#2-/*#)$&3*(+""&#$&*4+&'/-#2-&05"*+(6

Emma Timmins-Schiffman (UW, SAFS) Brook Nunn (UW, Med. Chem.)

David Goodlett (UW, Med. Chem.) Gary Dickinson (The College of NJ)

Steven Roberts (UW, SAFS)

!"#$$%$&"'#()*)#$+•! Important aquaculture species in the PNW and

worldwide •! Ecological services: water filtration, habitat, food

0-+/$&1-#.#2-/*#)$&/$.&7#8/98+"6

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 6

2848 µatm

638 µatm

1182 µatm

427 µatm

810 µatm 991 µatm

:;<+(#,+$*/9&=+"#%$6pCO2 (!atm)

400 600 800 1000 1200 2800

1 month exposure

t0: shell weight

No additional stress

Mechanical stress

No additional Mechanical Mechanical

•! Shell weight •! Gill tissue:

•! Transcriptomics •! Proteomics

•! Whole body: fatty acids

Shell weight

Heat shock •! 2 sublethal

temperatures: 42 & 43°C

•! 1 lethal temperature: 44°C

Mechanical Mechanical Mechanical Heat shock

:;<+(#,+$*/9&=+"#%$6pCO2 (!atm)

400 600 800 1000 1200 2800

1 month exposure

No additional stress

Mechanical stress

No additional Mechanical Mechanical Mechanical

•! Shell weight •! Gill tissue:

•! Transcriptomics

•! Proteomics •! Whole body: fatty acids

Shell weight

:;<+(#,+$*/9&=+"#%$6•! Mechanical stress as a proxy for environmental

stress •! MS promotes a catecholmine stress response in C.

gigas (Lacoste et al. 2001)

Proteins? Proteins? Proteins?

>+*4)."?&'()*+),#-"6•! Expression of genes and proteins change in

response to the environment •! Shotgun techniques provide non-biased surveys of

molecular physiological changes Pr

ote

in E

xpre

ssio

n

Normal environment Stressful environment

>+*4)."?&'()*+),#-"6•! 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


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


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

m/zm/z

95

100 1661.2975

1424.5980

1490.5931

1912.43041912.43041001912.43041001607.7299

1851.95781851.95781851.9578 10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

1001344.03811221.0632

1178.4297

1039.4359

1424.5980

1490.5931

1912.4304

1607.7299869.9010545.8882 823.7305

479.22881851.9578

Matched to “spectrum” in database

@+"A9*"?&'()*+),#-"6

•! 2,677 proteins were identified

•! Coverage of entire C. gigas proteome: 9.5%

@+"A9*"?&'()*+),#-"6

Transcriptional processes are the most significantly enriched process

-0.005 0.000 0.005 0.010

-0.005

0.000

0.005

Axis 1

Axi

s 2

High1

High2

High3

High4

Low1

Low2

Low3Low4

<B0C60.000

0.005

Low1

Low2

Low3Low4

High1

High2

High3

High4High2High4High2

pCO2 has an effect on the proteome

<B0C6pCO2 significantly affects shell hardness

-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

Low1

Low2

Low3

Low4

LowMS1

LowMS2

LowMS3

LowMS4

>+-4/$#-/9&3*(+""6

-0.010

-0.005

0.000

-0.005

0.000

Low1

Low2

Low3

Low4

LowMS1

LowMS2

LowMS3

LowMS4

-0.01 0.00 0.01 0.02

-0.02

-0.01

0.00

0.01

0.02

Axis 1

Axi

s 2

High1High2

High3High4

HighMS1

HighMS2

HighMS3

HighMS4

>+-4/$#-/9&3*(+""6

HighMS1

HighMS2

HighMS3HighMS3

HighMS4

High3

High1High2

High3High4

There is less of a proteomic response to a second stressor at high pCO2

>+-4/$#-/9&3*(+""6

,'--./(01*2'"%$(3445+

<B0C&/$.&>+-4/$#-/9&3*(+""6

•! Stress Response o! Heat shock protein 70 o! Caspase

•! pH Homeostasis •! V-type proton ATPase

•! Immune Response o! Cathepsin B o! MAP kinase kinase

3A,,/(56Proteomics can provide insight into the physiological response to ocean acidification.

3A,,/(56Exposure to multiple stressors can impact an organism’s ability to mount a successful response to either stressor.

D,<9#-/*#)$"6We 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

1-E$)F9+.%+,+$*"6•! 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 •! Funding: NOAA Saltonstall-Kennedy, RocketHub

1-E$)F9+.%+,+$*"6

0-+/$& 1-#.#2-/*#)$&3*(+&$&...

Documents

0-+/$& 1-#.#2-/#)$&3(+&$&...