Transcript Proteomic Assessment of Thiol Modifications Victor Darley-Usmar, Ph.D. Center for Free Radical Biology, University of Alabama at Birmingham.

Proteomic Assessment of Thiol
Modifications
Victor Darley-Usmar, Ph.D.
Center for Free Radical Biology, University of Alabama at
Birmingham
Increased protein modification in
cell signaling or oxidative stress
ROS/RNS
nitrotyrosine
thiol modification
carbonyl formation
Modified proteins
(altered function)
Proteomics is the study of a
protein complement in response to
a stimulus
Potential for biomarkers
Defining mechanisms
Hypothesis Generation
Some Reactive Proteomes In Free
Radical Biology
Thiol
Nitro
Carbonyl
Electrophile
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 MA
Nat Chem Biol. 2005 Aug;1(3):154-8. Epub 2005 Jul 10.
Sub-Classes of the Thiol Proteome
–S–S–R
–SNO
–SH
–SH
RSH
–SNO
–S-
HS–
ROS
ROS
–SH
O
–S
–SOH
H
OH
–S
–SH
–SR
Cooper et al. Trends Biochem. Sci. 2002
–S–OH
–S–OH
–SH
–SH
=O
–SH
=O
ONOO-
–S-
–SH
=O
=O
NO, RNS
–S–S–
Modifications Discussed
–S–S–R
–SNO
–SH
–SH
RSH
NO, RNS
–S-
–S–S–
–SH
HS–
–SROS
O
–SR
–SH
–SX
–SOH
–SX
–SH
Step 1: Are thiols modified at all?
tag
–S-
–S-tag
Western blot/
ROS/RNS
–SX
signal
Imaging
signal
Biotin as a tag
Advantages
Wide range of commercially
synthesized tags available.
extremely sensitive when coupled
with streptavidin/HRP
N-(biotinoyl)-N'- (iodoacetyl)ethylenediamine
(BIAM)
Can be used to pull down targets
Can be quantitative
Less sensitive to local protein
environment (c.f. antibodies)
Biotin as a tag
BIAM
BIAM
bt-15d-PGJ2
Disadvantage:
Endogenous carboxylases
105K
75K
N-(biotinoyl)-N'- (iodoacetyl)ethylenediamine
(BIAM)
Biochem J 394:185-95 (2006)
Mitochondria
Cells
Cytochrome c as an internal standard for
protein and Biotin
Cytochrome c: small
(12,000 Da), water
soluble,
multiple surface
lysine residues.
Biotin Tagging through Lysine:
Band Density (Arbitrary Units)
Native Cytochrome c - 12360
Matrix Adduct - 12569
Biotin (pmol) 9
3000
18 88 175
bt cyt.c
2500
2000
1500
1000
500
0
0
20 40 60 80 100 120 140 160 180
Biotin (pmol)
3 Biotins - 13374
4 Biotins - 13713
5 Biotins - 14052
2 Biotins - 13034
6 Biotins - 14391
7 Biotins - 14731
Apomyoglobin Standard
8 Biotins - 15068 16952
1 Biotin, 1 K 12733
10000.0
12000
14000
Mass (m/z)
Free Radic Biol Med. 40(3):459-68 (2006)
16000
18000
20000
Step 1:Prepare the sample and analyze by
1D-SDS-PAGE
Treatment
lyse sample with
BIAM at pH 8.0-8.5
N-(biotinoyl)-N'- (iodoacetyl)ethylenediamine
(BIAM)
Anal. Biochem. 283:214-221, 2000
Biochem J 379:359-366, 2004
detect biotin
(Western)
Step 2: Application to a 2D-Proteomic Format
(Rat Liver Mitochondria)
Sypro Ruby stain
biotin blot
Thiol Proteome
Abundance
Protein amt x dye binding Protein Amt x SH groups x
reactivity
Biotin tag is more sensitive than the
Sypro Stain
-bt
biotin
protein
0.3μg
0.01μg
abundance proteome is not the same as thiol proteome
S-Bt
S-Bt
–S–S–R
–SNO
–SH
–SH
RSH
NO, RNS
–S-
–S–S–
–SH
HS–
–SROS
O
–SR
–SH
–SX
–SOH
–SX
–SH
Diagonal electrophoresis for inter-protein disulfides
S S
hi
SH
oxidative
stress
excise
lane
SH
Reduce
low
S
Identify proteins off of diagonal
S
N-terminal Edman degradation
sequencing
Mass spectrometry
Immunoblot and probe for candidate
proteins
Adapted from
S S
S
S
J Biol Chem. 2004 Oct 1;279(40):41352-60.
–S–S–R
–SNO
–SH
–SH
GSH
Cys
NO, RNS
–S-
–S–S–
–SH
HS–
–SROS
O
–SR
–SH
–SX
–SOH
–SX
–SH
protein S-
HS-X -biotin
oxidizing
environment
GSH
X = GSH ester
Cys
protein S-S-X-biotin
detection, purification, imaging,
identification using
avidin-based methodologies
Protein
Biotin
–S–S–R
–SNO
–SH
–SH
RSH
NO, RNS
–S-
–S–S–
–SH
HS–
–SROS
O
–SR
–SH
–SX
–SOH
–SX
–SH
Differences in structure due to PTM of SH group
in Biology are subtle
Sulfenic
S-nitrosothiol
S
N
Sulfinic
RSOH
O
RSO2H
Surrounding amino residues will lead to epitope bias
SNO
-S
protein SOH
S-nitrosothiol
Sulfenic acid
Strategies
Direct 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.
-S
protein SOH
Sulfenic Acid
Strategies
Direct detection of the PTM.
Dimedone
protein
protein
SOH
Does not react with thiol, sulfinic, sulfonic, disulfide,
GSNO, Met Sulfoxide groups.
SNO
-S
protein SOH
Strategies
Differential chemical properties leading to specific
insertion of a tag.
BIOTIN SWITCH
SNO
-S
protein SOH
Alkylation to block free S-
Remove alkylating
agent
SNO
RS protein SOH
Restore the SOH or SNO to S-
R-S protein SOH
arsenite
reduction
R-S protein SNO
Remove reagents
ascorbate
reduction
TAG
R-S protein S-BT
R-S protein SBT
AFFINITY PURIFY and DETECT
Examples of RSNO/RSOH
Biotin
Protein
RSNO in endotoxin trtd
macrophage
Biotin
RSOH in peroxide (100 mM)
treated heart
How abundant are S-nitrosated Proteins?
DetaNONOate
Lyse and treat
cells (BAEC)
with BIAM
2D-IEF
detect biotin
Reactive Thiols
Protein stained gel
pH
Control-SH Blot
3
pH
After NO treatment-SH Blot
10
3
pH
10
150
100
75
50
35
30
15
10
Master map
Total spots = 135
Matched =41
Matched
Unmatched
PNAS. 2004:101(1):384-9
70% thiols
modified
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%
The problem of false positives
30% SX PTM in a population of 20 proteins
Block
93%effic.
S- SR SX STag
False Positive
is 14%
Convert
Tag
The problem of false positives
5% SX PTM in a population of 20 proteins
S- SR SX STag
Block
93%effic.
Convert
False Positive
is 50%.
Tag
Detecting Specific Modifications
–S–S–R
–SNO
–SH
–SH
RSH
NO, RNS
–S-
–S–S–
–SH
HS–
–SROS
O
–SR
–SH
–SX
–SOH
–SX
–SH
Future Directions; organelle specific
–SH
H+ H+ +
H
+
H
+ H+
H
H+
+
H+ H H+
eO2
+
H
ATP
ADP
S–TPP
–
IgG
P
IBTP+
H+
+
I
2D SDS-PAGE followed by western blotting
Control
Anti-IBTP
Ethanol
HSP70
1
2
3
5
1
2
3
4
5
6
7
6
4
Aldehyde
dehydrogenase
7
Mass MOWSE No. peptides matched/
unmatched
(kDA) score
Pyruvate carboxylase
129.6 195
28/49
Hsp70
72.1
194
28/74
Hsp60
60.9
90
8/13
Glutamate dehydrogenase
56
98
8/15
Protein disulfide isomerase
56.9
123
10/9
Mitochondrial aldehyde dehydrogenase 53
135
12/15
Acetyl-coenyzme A acyl transferase 2 41.8
79
7/13
.
Am J Physiol Gastrointest Liver Physiol. 2004 Apr;286(4):G521-7
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
Current Lab Members
Elena Ulasova
Joo-Yeun Oh
Jessica Gutierrez
Brian Dranka
Balu Chacko
Ashlee Preston
Jeff Dubuisson
Former Members
Nobuo Watanabe
Jaroslaw Zmijewski
Claire Le Goffe
Niroshini Giles
Anna-Liisa Levonen
Sruti Shiva
Collaborators
Aimee Landar
Anne Diers
Yeun Su Choo
Karina Ricart
Michelle Johnson
Stephen Barnes
Paul Brookes
Dale Dickinson
Jason Morrow
Lewis Pannell
Shannon Bailey
Neil Hogg
Scott Ballinger
Philip Eaton
Bruce King
Selected References for Thiol Proteomics
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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.