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2
Presented at the 2011 Stanford Mass Spectrometry Users’ Meeting
Healthy CarcinomaCirrhosis
C57B6 female micemice
(6 mo.)
ildWild type (WT)
Sod1 Sod1 Sod1
HCV‐S
Sod1 Sod1 Sod1
HCV‐FL
Sod1 Sod1 Sod1Sod1
+/+
Sod1
‐/+
Sod1
‐/‐
Sod1
+/+
Sod1
‐/+
Sod1
‐/‐
Sod1
+/+
Sod1
‐/+
Sod1
‐/‐
Core E1 E2 p7 HCV-S
Core E1 E2 p7 NS3 NS4A NS4B NS5A NS5B HCV-FL
Treatments:
Control vs Alcohol Biochemical ProteomicsControl vs. Alcohol diets
analyses Proteomics
Treatments:
Control vs.
Biochemical analyses:
Liver enzyme activity; ProteomicsAlcohol
diet
Liver enzyme activity;
Histopathology; etc…
Proteomics
Proteomics:
Treatments:
Control vs Alcohol
Biochemical analyses:
Liver enzyme activity;
Detecting protein modification via
bisANS or disulfide Control vs. Alcohol
diet
Liver enzyme activity;
HistopathologybisANS or disulfide
labeling;
2D electrophoresis; Mass Spectrometry
15001400 -Control14001300
1200110010001000
900800
700600600500
400300200200100
0+/+ -/+ -/- +/+ -/+ -/- +/+ -/+ -/-
WT HCV-S HCV-FL
15001400 - Control14001300
120011001000
- Alcohol
1000
900800
700600600500
400300200200100
0+/+ -/+ -/- +/+ -/+ -/- +/+ -/+ -/-
WT HCV-S HCV-FL
Control
Sod1 +/+ Sod1 -/+ Sod1 -/-Normal liver
Alcohol
Sod1 +/+ Sod1 -/+ Sod1 -/-Fatty liver
1 BisANS for protein conformational changes1. BisANS for protein conformational changes
1. 6‐IAF for protein modification
2-D gel electrophoresiAdd bisANS UV irradiation
sAdd bisANS U ad a o
& cross linkProtein extraction
SH
-SH
-S
ReducingAdd IAAProtein extraction
Reducing agent
Add IAA
-SH
-SH
2-D gel electrophoresi
sAdd 6-IAF
s
Total proteinvia
Coomassie blue
Hydrophobicityvia
bisANS
Disulfide bondvia
6-IAF
LC/MSD Trap XCT Ultra Agilent TechnologiesLC/MSD Trap XCT Ultra, Agilent Technologies
CytoplasmMetal-bindingPhosphoproteinAcetylationAlcohol catabolic processAlcohol catabolic processGlycolysis/GluconeogenesisMitochondrionMitochondrial lumen & matrixHomeostatic processAntioxidantOxidation reductionResponse to oxidative stressResponse to ROS
Response to H2O2Response to H2O2
Neg. regulation of apoptosis & death
DAVID Bioinformatics Resources, NIAID
CytoplasmMetal-bindingPhosphoproteinAcetylationAlcohol catabolic processAlcohol catabolic processGlycolysis/GluconeogenesisMitochondrionMitochondrial lumen & matrixHomeostatic processAntioxidantOxidation reductionResponse to oxidative stressResponse to ROS
Response to H2O2Response to H2O2
Neg. regulation of apoptosis & death
DAVID Bioinformatics Resources, NIAID
CytoplasmMetal-bindingPhosphoproteinAcetylationAlcohol catabolic processAlcohol catabolic processGlycolysis/GluconeogenesisMitochondrionMitochondrial lumen & matrixHomeostatic processAntioxidantOxidation reductionResponse to oxidative stressResponse to ROS
Response to H2O2Response to H2O2
Neg. regulation of apoptosis & death
DAVID Bioinformatics Resources, NIAID
CytoplasmMetal-bindingPhosphoproteinAcetylationAlcohol catabolic processAlcohol catabolic processGlycolysis/GluconeogenesisMitochondrionMitochondrial lumen & matrixHomeostatic processAntioxidantOxidation reductionResponse to oxidative stressResponse to ROS
Response to H2O2Response to H2O2
Neg. regulation of apoptosis & death
DAVID Bioinformatics Resources, NIAID
CytoplasmMetal-bindingPhosphoproteinAcetylationAlcohol catabolic processAlcohol catabolic processGlycolysis/GluconeogenesisMitochondrionMitochondrial lumen & matrixHomeostatic processAntioxidantOxidation reductionResponse to oxidative stressResponse to ROS
Response to H2O2Response to H2O2
Neg. regulation of apoptosis & death
DAVID Bioinformatics Resources, NIAID
CytoplasmMetal-bindingPhosphoproteinAcetylationAlcohol catabolic processAlcohol catabolic processGlycolysis/GluconeogenesisMitochondrionMitochondrial lumen & matrixHomeostatic processAntioxidantOxidation reductionResponse to oxidative stressResponse to ROS
Response to H2O2Response to H2O2
Neg. regulation of apoptosis & death
DAVID Bioinformatics Resources, NIAID