proteomic assessment of thiol modifications victor darley-usmar, ph.d
DESCRIPTION
Proteomic Assessment of Thiol Modifications Victor Darley-Usmar, Ph.D. Center for Free Radical Biology, University of Alabama at Birmingham. nitrotyrosine. thiol modification. carbonyl formation. Increased protein modification in cell signaling or oxidative stress. ROS/RNS. - PowerPoint PPT PresentationTRANSCRIPT
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Proteomic Assessment of Thiol Modifications
Victor Darley-Usmar, Ph.D.Center for Free Radical Biology, University of Alabama at
Birmingham
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Increased protein modification in
cell signaling or oxidative stress
ROS/RNS
Modified proteins (altered function)
nitrotyrosinethiol modification
carbonyl formation
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Proteomics is the study of a protein complement in response to a stimulus
Potential for biomarkers Defining mechanisms
Hypothesis Generation
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Some Reactive Proteomes In Free Radical Biology
Thiol
Nitro Carbonyl
Electrophile
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Role of thiols in protein function and cell signaling
“redox signaling”
Thioredoxin catalyzes the S-nitrosation of the caspase-3 active site cysteine.Mitchell DA, Marletta MANat Chem Biol. 2005 Aug;1(3):154-8. Epub 2005 Jul 10.
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–S-
–S-
RSH
–SH
–S–S–R
–S
–S
–SNO
–SH
NO, RNS
–S–S–
–SH
HS–
=O
–SH
ONOO-
–SNO ROS
ROS
=O
–S–OH
=O
–SOH
–SH
–SH
=O
–S–OH
–SH
Cooper et al. Trends Biochem. Sci. 2002
–SH
–SR
H
O
OH
Sub-Classes of the Thiol Proteome
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–S-
–S-
RSH
–SH
–S–S–R
–SNO
–SH
NO, RNS
–S–S–
–SH
HS–
ROS
–SOH
–SH–SH
–SR
Modifications Discussed
O
–SX
–SX
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–S- tag –S-tag
signal
Western blot/
Imaging
–SX
ROS/RNS
signal
Step 1: Are thiols modified at all?
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Biotin as a tag
N-(biotinoyl)-N'- (iodoacetyl)-
ethylenediamine(BIAM)
Advantages
Wide range of commercially synthesized tags available.
extremely sensitive when coupled with streptavidin/HRP
Can be used to pull down targets
Can be quantitative
Less sensitive to local protein environment (c.f. antibodies)
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Biotin as a tag
N-(biotinoyl)-N'- (iodoacetyl)-
ethylenediamine(BIAM)
Disadvantage:Endogenous carboxylases
105K
75K
b
t-15
d-P
GJ 2
Biochem J 394:185-95 (2006) MitochondriaB
IAM
Cells
BIA
M
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Cytochrome c: small (12,000 Da), water soluble, multiple surface lysine residues.
Cytochrome c as an internal standard for protein and Biotin
Biotin Tagging through Lysine:
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Native Cytochrome c - 12360
Matrix Adduct - 12569
10000.0 12000 14000 16000 18000 20000Mass (m/z)
3 Biotins - 13374
4 Biotins - 13713
5 Biotins - 14052
2 Biotins - 13034
6 Biotins - 14391
7 Biotins - 14731 Apomyoglobin Standard169528 Biotins - 150681 Biotin, 1 K -
12733
bt cyt.c
17588189Biotin (pmol)
0 20 40 60 80 1001201401601800
500
1000
1500
2000
2500
3000
Ban
d D
en
sit
y (
Arb
itra
ry U
nit
s)
Biotin (pmol)
Free Radic Biol Med. 40(3):459-68 (2006)
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N-(biotinoyl)-N'- (iodoacetyl)-ethylenediamine
(BIAM)
Anal. Biochem. 283:214-221, 2000
Step 1:Prepare the sample and analyze by 1D-SDS-PAGE
detect biotin (Western)
Treatment
lyse sample with BIAM at pH 8.0-8.5
Biochem J 379:359-366, 2004
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Sypro Ruby stain biotin blot
Step 2: Application to a 2D-Proteomic Format
(Rat Liver Mitochondria)
AbundanceProtein amt x dye binding
Thiol ProteomeProtein Amt x SH groups x reactivity
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Biotin tag is more sensitive than the Sypro Stain
-bt-bt
bt-
protein biotin
0.3μg 0.01μg
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abundance proteome is not the same as thiol proteome
S-Bt
S-BttB-S
S-B
t
S-Bt
S-B
t
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–S-
–S-
RSH
–SH
–S–S–R
–SNO
–SH
NO, RNS
–S–S–
–SH
HS–
ROS
–SOH
–SH–SH
–SR
O
–SX
–SX
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Diagonal electrophoresis for inter-protein disulfides
hi
low
SH
SH
S S
oxidativestress
exciselane
Identify proteins off of diagonal
N-terminal Edman degradation sequencing
Mass spectrometry
Immunoblot and probe for candidate proteins
S S
S
S
Reduce
S
S
Adapted from J Biol Chem. 2004 Oct 1;279(40):41352-60.
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–S-
–S-
GSHCys
–SH
–S–S–R
–SNO
–SH
NO, RNS
–S–S–
–SH
HS–
ROS
–SOH
–SH–SH
–SR
O
–SX
–SX
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-biotin protein S- HS-X
protein S-
oxidizingenvironment
-biotinS-X
detection, purification, imaging, identification using
avidin-based methodologies
GSHGSH esterCys
X =
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Protein Biotin
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–S-
–S-
RSH
–SH
–S–S–R
–SNO
–SH
NO, RNS
–S–S–
–SH
HS–
ROS
–SOH
–SH–SH
–SR
O
–SX
–SX
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Differences in structure due to PTM of SH group in Biology are subtle
Surrounding amino residues will lead to epitope bias
S
N O
RSOH
RSO2H
S-nitrosothiol Sulfenic Sulfinic
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StrategiesDirect detection of the PTM.
Antibody: epitope too small and not structurally distinct. Mass Spectrometry: Sensitivity often not adequate
Differential chemical properties leading to specific insertion of a tag.
protein
SNO
SOH-S Sulfenic acid
S-nitrosothiol
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StrategiesDirect detection of the PTM.
protein SOH-S
proteinSOH
protein
Does not react with thiol, sulfinic, sulfonic, disulfide,GSNO, Met Sulfoxide groups.
Sulfenic Acid
Dimedone
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Strategies
Differential chemical properties leading to specificinsertion of a tag.
protein
SNO
SOH-S
BIOTIN SWITCH
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protein
SNO
SOH-S
Alkylation to block free S-
Remove alkylatingagent
protein
SNO
SOHRS
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ascorbate reduction
protein SNO protein SOHR-S R-S
arsenite reduction
Remove reagents
Restore the SOH or SNO to S-
protein SBT protein S-BTR-S R-S
TAG
AFFINITY PURIFY and DETECT
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Examples of RSNO/RSOH
RSNO in endotoxin trtdmacrophage
Biotin Protein
RSOH in peroxide (100 M)treated heart
Biotin
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pHProtein stained gel
detect biotin Reactive Thiols
Lyse and treat cells (BAEC)
with BIAM
DetaNONOate
2D-IEF
How abundant are S-nitrosated Proteins?
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150
10075
50
35
30
15
10
3 10pH
3 10pH
Control-SH Blot After NO treatment-SH Blot
Master map
Total spots = 135Matched =41
Matched
Unmatched
PNAS. 2004:101(1):384-9
70% thiolsmodified
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Measure RSNO and thiols by direct non-proteomics
technique.
RSNO 11.2 ± 0.07pmol/mg protein
Protein Thiol approx 40-80 nmol/mg protein
0.014-0.028%
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The problem of false positives
S- SR SX
30% SX PTM in a population of 20 proteins
Convert Tag
STag
False Positiveis 14%
Block 93%effic.
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The problem of false positives
S- SR SX
5% SX PTM in a population of 20 proteins
Convert Tag
STag
False Positiveis 50%.
Block 93%effic.
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–S-
–S-
RSH
–SH
–S–S–R
–SNO
–SH
NO, RNS
–S–S–
–SH
HS–
ROS
–SOH
–SH–SH
–SR
Detecting Specific Modifications
O
–SX
–SX
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ADPATP
H+
H+e- O2
H+
H+
H+H+
H+
H+H+
H+H+ H+
Future Directions; organelle specific
P I+
IBTP+
–SH
–S–TPP
IgG
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Control EthanolAnti-IBTP
Aldehyde dehydrogenase
HSP70
2D SDS-PAGE followed by western blotting
1 Pyruvate carboxylase 129.6 195 28/492 Hsp70 72.1 194 28/743 Hsp60 60.9 90 8/134 Glutamate dehydrogenase 56 98 8/155 Protein disulfide isomerase 56.9 123 10/96 Mitochondrial aldehyde dehydrogenase 53 135 12/157 Acetyl-coenyzme A acyl transferase 2 41.8 79 7/13
Mass (kDA)
MOWSE score
No. peptides matched/unmatched
12
34
56 7
Am J Physiol Gastrointest Liver Physiol. 2004 Apr;286(4):G521-7.
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Challenges
Matching the proteome with tag pattern
Developing internal standard for gel and blot
Secondary reactions may also lead to thiol Modification
Thiol proteomes are composed of discreet low abundance proteins
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Current Lab MembersAimee Landar
Anne DiersYeun Su ChooKarina Ricart
Michelle Johnson Stephen Barnes
Paul BrookesDale Dickinson Jason MorrowLewis Pannell
Shannon BaileyNeil Hogg
Scott Ballinger
Philip EatonBruce King
Elena UlasovaJoo-Yeun Oh
Jessica GutierrezBrian DrankaBalu Chacko
Ashlee PrestonJeff Dubuisson
Former MembersNobuo Watanabe
Jaroslaw Zmijewski Claire Le Goffe Niroshini Giles
Anna-Liisa LevonenSruti Shiva
Collaborators
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Selected References for Thiol
Proteomics • Eaton, P. (2006) Protein thiol oxidation in health and disease: techniques for measuring disulfides and related
modifications in complex protein mixtures. Free Radic Biol Med 40, 1889-1899• Good overview of the various methods available for measuring thiol redox status in a proteomics context and the
principles involved. • Poole, L. B., Zeng, B. B., Knaggs, S. A., Yakubu, M. and King, S. B. (2005) Synthesis of chemical probes to map sulfenic
acid modifications on proteins. Bioconjug Chem 16, 1624-16028.• Example of the strategies to develop a thiol tag that can be applied to proteomics.• Landar, A., Oh, J. Y., Giles, N. M., Isom, A., Kirk, M., Barnes, S. and Darley-Usmar, V. M. (2006) A sensitive method for the
quantitative measurement of protein thiol modification in response to oxidative stress. Free Radic Biol Med 40, 459-468• Method for the quantitative measurement of biotin tags in proteomics gel formats. • Patton, W. F. (2002) Detection technologies in proteome analysis. J Chromatogr B Analyt Technol Biomed Life Sci 771, 3-
31• Broad overview of the various approaches to assessing post-translational modification of proteomes. • Gao, C., Guo, H., Wei, J., Mi, Z., Wai, P. Y. and Kuo, P. C. (2005) Identification of S-nitrosylated proteins in endotoxin-
stimulated RAW264.7 murine macrophages. Nitric Oxide 12, 121-126.• An application of the biotin switch method as applied to S-nitrosothiols showing endogenous protein S-nitrosation. • Gladwin, M. T., Wang, X. and Hogg, N. (2006) Methodological vexation about thiol oxidation versus S-nitrosation -- a
commentary on "An ascorbate-dependent artifact that interferes with the interpretation of the biotin-switch assay". Free Radic Biol Med 41, 557-561
• Discussion of the problem of false positives in biotin switch methods.• Dennehy, M. K., Richards, K. A., Wernke, G. R., Shyr, Y. and Liebler, D. C. (2006) Cytosolic and nuclear protein targets of
thiol-reactive electrophiles. Chem Res Toxicol 19, 20-29• Use of mass spectrometry proteomics analysis to define the electrophile responsive proteome in cells. • Levonen, A. L., Landar, A., Ramachandran, A., Ceaser, E. K., Dickinson, D. A., Zanoni, G., Morrow, J. D. and Darley-
Usmar, V. M. (2004) Cellular mechanisms of redox cell signalling: role of cysteine modification in controlling antioxidant defences in response to electrophilic lipid oxidation products. Biochem J 378, 373-382
• An example of the candidate protein approach using different tagging approaches to identify modification of a cell signaling molecule.