Reactive Thiols in Redox Signaling
SFRBM Worshop
Boston University
Medical Center
November 15,
BU-NHLBI Cardiovascular
2006 Proteomics Center
Denver, CO
Richard A. Cohen, MD
Director, Vascular Biology Unit
Boston University School of Medicine
[email protected]
Outline
• Introduction: Reactive thiol proteome
• MS screening for reactive thiols with ICAT
• S-glutathiolation labeling and detection
methods
• S-glutathiolation in signaling
Cysteine Thiols Modified by Oxidants
Constitute a Thiol Proteome
Pathological
Physiological
Reversible
Signaling
Irreversible
Damage
Pr-Cys-SH
Pr-Cys-SOH
Pr-Cys-SO3H
Pr-Cys-SO2H
Pr-Cys-S-SG
Pr-Cys-SNO
ROS
Reactive Cysteine
Thiolate Anion
H2O2
NO2 ONOO-
.OH
.
NO
SH
S-
O
O
SNO, SOxH
O
NH
OH
NH
NH
O
Cysteine
NH
Reactive Cysteine
Thiolate Anion
ROS/RNS
GSH
(0.5 - 5 mM)
SH
S
SS
SG
O
O
.
GS
GSOH
GSSG
GSNO
O
NH
OH
NH
NH
S-Glutathiolation:
NH
O
Cysteine
Large - tripeptide
Charged - glutamate
Reversible GRX,TRX
MS screening for reactive thiols with ICAT
Biotin-labeled Iodoacetamide:
Affinity tag
(Biotin)
Thiol-reactive
iodoacetamide
group
Reduced Reactive Thiols
Acid-Cleavable ICAT (Applied Biosystems, USA):
Affinity tag
(Biotin)
Acid-cleavable
linker
Isotopecoded tag
Light (9 12C)
Heavy (9 13C)
Thiol-reactive
iodoacetamide
group
Nature Biotechnology
1999, 17, 994-999
Aebersold, R. and
Coworkers
ICAT Approach to Redox Proteomics: Creatine Kinase
Non-reactive cysteine
Reactive cysteine
Labeling at pH= 8.5
Peptide with nonreactive cysteine 146
Normal
Light 9 -12C ICAT
%
H2O2
%
10%
light heavy
LC-MS
m/z
Peptide with
reactive cysteine 283
Oxidant stress
Heavy 9-13C ICAT
%
H2O2
90%
Ox
Ox
light heavy
Mix and trypsin digestion
Avidin separation
m/z
M. Sethuraman et al. Mol Cell Proteomics 3: 273, 2004
ICAT approach to complex protein mixtures
Sucrose
gradient
Isolated
rabbit heart
200 g protein
No H2O2
SR membrane fractionreducing agent/protease
inhibitor free, 50 mM Tris
pH 7.1
Light ICAT
200 g protein,
10 mM H2O2
30 mins/RT
Heavy ICAT
Mixed, Trypsin
digestion, Avidin
affinity and LCMS/MS
M. Sethuraman et al. J. Proteome Research, 2004, 3(6):1228-33.
Total Ion Chromatogram for LC-ESI-MS/MS of ICAT-labeled Tryptic
Peptides from HPLC Cation Exchange Fraction 2 of Membrane Proteins
24.89
100
29.00
31.38
35.70
16.34 17.86
%
39.18
21.60
52.14
13.57
44.87
46.81
54.47
0
10.00
20.00
Time in minutes
30.00
40.00
50.00
MS of ions eluted at 29.002 min
100
Val232-Arg245 of GAPDH
576.319
579.325
576.651
%
533.318
100
0
579.659
576.984
577.307
579.992
580.316
577
578
579
580
m/z
515.306
%
371.717
736.395
981.507
561.317 671.357
773.430863.979900.471
570.651
999.469
0
m/z
350
450
550
650
750
850
950
1050
1150
MS/MS Sequenced ICAT-labeled Peptides in Cardiac Membrane Fraction
Ladder
Score
Name
Description
KCRS_RABIT
Creatine kinase sarcomeric mitochondrial precursor
721.08phosphate dehydrogenase
Glyceraldehyde
720.74
G3P_RABIT
100
Fructose biphosphate aldolase
a
%
723
721
725
Polymeric immunoglobulin receptor precursor
632.67 633.00
KPBB_RABIT
b
182 (R)GLSLPPACSR(A)
272
287 (R)LGYILTCPSNLGTGLR(A)
42.3
9.8
52.941
272
287 (R)LGYILTCPSNLGTGLR(A)
40.5
69.863
232
245 (R)VPTPNVSVVDLTCR(L)
26.9
64.835
143
159 (K)IVSNASCTTNCLAPLAK(V)
72.2
49.485
69
ICAT ICAT
86 (R)VNPCIGGVILFHETLYQK(A)
12.3
dehydrogenase
• 60 Cysteines ID’d by LC MS/MS
Sarcoplasmic endoplasmic reticulum Ca2+ ATPase
0
100
173
201
207 (R)CQYVTEK(V)
726.7683.871727.10
IVSNASCTTNCLAPLAK 24.3
56.364
331
341 (R)ALANSLACQGK(Y)
24.1
727.43
Glyceraldehyde-3-phosphate
50.633
437
451
(K)VGEATETALTCLVEK(M)
32.5
727.94
721.76
ATA2_RABIT
PIGR_RABIT
100
55.294
72%
721.42
ALFA_RABIT
% Cys
Oxidation
with H2O2
Start End Sequence
%
K6PF_RABIT
397 (K)VDGDTCSLNEFTITGSTYAPIGEVHK(D)
15.1
468
476
(R)ANACNSVIK(Q)
m/z
28.6
25.62
353
374 (R)SPPVLKGFPGGSVTIRCPYNPK(R)
16.279
587
595 (K)ARCPVPRRR(Q)
62%
• 9 Oxidized by >50%
Phosphorylase B kinase beta regulatory chain
633.65
Phosphofructose kinase muscle type
Voltage
633dependent anion selective channel protein
635 1
POR2_RABIT
750.91
Voltage-dependent anion-selective channel protein 2
751.42
100
372
79.07
727
36.082
633.34
0
POR1_RABIT
POR3_RABIT
44.138
• 3 Oxidized >70%
Voltage-dependent anion-selective channel protein 3
120
30.1
66.019
46
48%
96.364
Vitamin K dependent protein S precurson fragment
32.727
DHSA_HUMAN
NUFM_RAT
ODO1_HUMAN
Succinate dehydrogenase mitochondrial pre
NADH-ubiquinone oxidoreductase
64
28.767
64
755.42
57.143
172
755.93 756.42
75.676
753
47.273
755
kinase<5
66.99
Hemoglobin beta 1 chain
Malate dehydrogenase
62.3
(R)GITNLCVIGGDGSLTGADTFR(S)
Phosphorylase 
45.6
cHBB_RABIT%
751
(K)NIIYYKCNTHDER(E)
224637235
(K)YQIDPDACFSAK(V)
m/z
Plasma retinol binding protein
MDHM_RAT0
<5
NIIYYKCNTHDER
630
Phosphatidylcholine-sterol acyltransferase [Precursor]
PRTS_RABIT
13.7
(R)QTLCSLLPRESR(S)
81.967
RETB_RABIT
FA10_RABIT
ICAT
612 (R)RQWYPLSRKLRTSCPEPR(L)
59.223
LCAT_RABIT
751.92
Coagulation factor 752.41
595
635.68
29.508
276
287
636.01
32.836
832
844
636.35
59.13 636.68
108
128
24
99.6
50.746
334
756.92
88
442
92
757
648 (K)CNENYTTDFIFNLYSEEGK(G)
138 (R)EHINLGCDVDFDIAGPSIR(G)
64 (K)SCSGVEFSTSGSSNTDTGK(V)
48
54.38
74 (K)VCNYGLTFTQK(W)
48.6
77 (K)LDKPSVVNWMCYRK(T)
43.9
ICAT
181 (R)QRQEELCLSR(Q)
VCNYGLTFTQK
344 (R)RNVAPACLPQK(D)
95 (K)LSELHCDK(L)
28.1
39
49.6
Voltage-dependent anion
89.6
channel protein 3
104 (K)GCDVVVIPAGVPR(K)
26
452 (K)QKKHCLVTVEK(G)
m/z
64.384
233
246 (R)GVIALCIEDGSIHR(I)
26.7
56.164
233
246 (R)GVIALCIEDGSIHR(I)
26.7
64.706
7
22 (K)TTGLVGLAVCDTPHER(L)
Sethuraman 46.575
et al.278
J. Proteome
Res, Dec 2004
2-oxoglutarate dehydrogenase E1 component, mitochondrial
291 (R)FGLEGCEVLIPALK(T)
31.5
8.5
Reversible ICAT labeling due to S-glutathiolation of
recombinant p21ras Cys-118
Val103 –Arg123
a)
GSSG (0 μM) +
5 mM TCEP
b)
GSSG (0 μM)
830.003+
GSSG (100 μM)
GSSG (100 μM)
+ 5 mM TCEP
830.003+
833.023+
100
100
%
%
833.023+
0
830
832
834
m/z
0
830
832
834
m/z
M. Sethuraman et al., submitted 2006
•S-glutathiolation labeling and detection methods
•S-glutathiolation in signaling
Proteomic Screening for S-Glutathiolated Proteins
-GSH + Protein-SH +ONOO-
-GSS-Protein +Trypsin
-GSS-Peptide
+DTT
Peptide-SH
Reactive thiols bind to GSH
in greatest abundance
Measured Mass Computed Mass
1387.9
1387.7
SERCA
MALDI or ESI
Mass spectrometry
-GSS- C674FARVEPSHKSK
Adachi et al. Nature Medicine,10,1200-1207, 2004
SERCA: Sarcoplasmic/endoplasmic reticulum
Ca 2+ ATPase Cysteine-674
Phosphorylation
Domain
B - Domain
P
(1)
Nucleotide - binding
Domain
Cys-674
D351
294
Y
C674
Hinge Domain
Stalk Domain
(997)
Cytosolic Domain
Transmembrane
Domain
M1 M2 M3 M4 M5 M6 M7 M8 M9 M10M11
Luminal Domain
674
AQRDACLNARCFARVEP
S-
Olesen C, et al. Science
306:2251-5, 2004
GSH Sepharose pull-down of intact SERCA via
Cys-674 in transfected HEK cells
Reactive thiols bind to
GSH in abundance!
Ctr
-SSG-
Wt
C674S
110kDa-
GSSSERCA
110kDa-
SERCA
ONOO- (20 M)
Adachi et al. Nature Medicine,10,1200-1207, 2004
Proteomic Screening for S-Glutathiolated Proteins
-GSH + Protein-SH +ONOO-
-GSS-Protein +Trypsin
-GSS-Peptide
+DTT
Peptide-SH
Reactive thiols bind to GSH
in greatest abundance
Measured Mass Computed Mass
1387.9
1387.7
SERCA
MALDI or ESI
Mass spectrometry
-GSS- C674FARVEPSHKSK
Peptide
masses
ONOO- -induced Glutathiolation of Proteins in
p21ras is aRabbit
Redox-Sensitive
Aortic HomogenateSignaling
Molecule
peptides Protein
MW
861.0504
1318.672 titin
Na-K-ATPase Alpha subunit
1359.659 PP2A subunitA, PR65 regulatory beta
L-Ca-channel
1638.793 MLCK
Na+ Channel alpha
1766.626 ROCK-1
1791.699 RYR3
2343.926 PP2A subunitA, PR65 regulatory beta
2383.89 IgG
Ras
nNOS
ROCK-1
2441.977 IgG
Ras
nNOS
2490.252 Myosin
Na-K-ATPase Alpha subunit
2705.113 Ras
L-Ca-channel
nNOS
RYR3
RYR3
TKR
Shc
Grb
Sos
Ras
Raf
MEKK
MEK1
ERK1/2
MEK4
JNK
110.05
114.3
60.97
149.73
125.7
225.73
158.33
550.32
60.97
11.71
20.91
160.85
158.33
11.71
20.91
160.85
PI3K
MEK6
114.3
20.91
149.73
160.85
550.32
550.32
P38
Proliferation
Hypertrophy
Measured Mass
Computed Mass
Error
Da
Da
ppm
1317.664
1317.664
1358.651
1358.651
1637.785
1637.785
1765.618
1790.691
2342.918
2382.882
2382.882
2382.882
2382.882
2440.969
2440.969
2440.969
2489.244
2489.244
2704.105
2704.105
2704.105
2704.105
2704.105
Rac
NADPH
oxidase
Akt
1317.707
1317.732
1358.64
1358.724
1637.771
1637.801
1765.833
1790.926
2343.222
2383.137
2383.166
2383.104
2383.154
2441.142
2441.171
2441.109
2489.179
2489.195
2704.297
2704.352
2704.335
2704.358
2704.16
Start
-33
-52
9
-53
8
-10
-122
-131
-130
-107
-119
-93
-114
-71
-83
-57
26
20
-71
-92
-85
-94
-21
552
525
334
1186
1049
1159
740
1956
494
37
168
920
447
37
168
920
109
673
102
18
829
3376
2086
End
Sequence
563 ATKTPVSDLRCK
535 ILAAQGCK
345 CVSSPHFQVAER
1196 RASFHLECLKR
1063 DLEVVEGSAARFDCK
1173 RFPCCQVSIESGKGK
754 QCSMLDVDLKQSQQK
1970 CPTTLKELISQTMIR
514 RKSELPQDPHTKKLEAHCR
59 GLEWIACIRGGGSGSTNYASWAK
188 QHKLRKLNPPDESGRGCMSCK
939 MREGDELCGQEEAFRTWAKK
465 DEMEQKCRTSNIKLDKIMK
59 GLEWIACIRGGGSGSTNYASWAK
188 QHKLRKLNPPDESGRGCMSCK
939 MREGDELCGQEEAFRTWAKK
129 YAAWMIYTYSGLFCVTVNPYK
694 ACVINGMQLKDMDPSELVEALR
126 VKDSDDVPMLNKCDVGNKCDLAAR
39 NCELDKNQRQCVEYALKARPLR
850 CALMEMRHPNSLQEERKSYKVR
3399 KMLPIGLNMCTPGDQELISLKSR
2106 SQIAFPKMVASCCRFLCYFCR
Cys-118
(Mallis RJ, Thomas JA,
Biochem J 2001)
IAA
IAA, MO
IAA
IAA
IAA
IAA
IAA
IAA, MO
IAA(4)
Techniques available for S-glutathiolation
detection in cells
• Biotin labeled glutathione ester
• Radiolabeled glutathione
• Anti-GSH antibody
• Mass Spectrometry
• In situ visualization of glutathione adducts
Biotinylated GSH Ester Labeling of
S-Glutathiolated Proteins in Intact Cells
b
GSH-ester
membrane
b
GSH
ROS
Pr-S-
b
GS-SPr
SA beads Western blot
Proteomic studies
Thiol reduction
Biotinylated GSH Ester Synthesis
50 mM NaHCO3
+
pH 8.5
Biotin Sulfo-NHS
2h, RT
50 mM NH4HCO3
pH 8.5
30 min, RT
+
Biotin Sulfo-NHS
GSH ethyl ester
--N-CO- Biotin
GSH ethyl ester
--N-CO- Biotin
--NH2
GSH ethyl ester
+
Biotin -CO-HNH
Protocol for Detection of Cellular S-glutathiolated
Proteins with Biotinylated GSH Ester
250 µM BGSH ester
1h incubation
Treatment X
X min incubation
Cell Lysis and elimination of low
MW components on PD-10 size
exclusion columns
Affinity Purification:
Streptavidin…
1h to overnight,
4°C
Washing step
5 times:
0.5 M NaCl 0.1% SDS
Protein
Recovery
10 mM DTT
SDS-PAGE
Biotinylated GSH Ester Tracer for
S-Glutathiolated Proteins
b
GSH-ester
GSS-Proteins in
Rat SMC
membrane
ROS
Pr-S-
b
GSH
b
GS-SPr
SA Pull-down
SA Blot
Adachi, JBC 2004
Cont Ang II Cont Ang II
-DTT +DTT(10 mM)
Streptavidin blot
b
GSH-ester
membrane
ROS
b
Tension, 10 g
NO S-Glutathiolates SERCA and Relaxes the Rabbit Aorta
PE
NO
1
(min)
Bio-GSE
1h
GSH
-bGSS-SERCA
110 kDa-
Pr-S-
30
b GS-SPr
0
1
30 (min )
NO (1 M)
SA Pull-down
Blot SERCA
Adachi et al. Nature Medicine,10,1200-1207, 2004
Decreased NO-induced S-Glutathiolation of
SERCA in Atherosclerotic Rabbits
-bGSS-SERCA
20
*
N.S.
(unit)
15
10
Tension, 10 g
-SERCA
PE
NO
1
Bio-GSE
1h
5
0
PE
PE+NO
Normal
PE
PE+NO
Atherosclerotic
Adachi et al. Nature Medicine,10,1200-1207, 2004
Biotinylated GSH Ester Tracer for
S-Glutathiolated Proteins
b
GSH-ester
membrane
ROS
Pr-S-
b
GSH
b
Ascorbate
DTT
ONOOIB
p21ras
-
+
+
+
+
+
--21
kDa
GS-SPr
SA Pull-down
Blot p21ras
Clavreul et al. FASEB J, 2006
p21ras S-Glutathiolation caused by
Angiotensin II in RASMC
b GSH-ester
Ras S-Glutathiolation
-GSS-Ras
membrane
-Ras
ROS
Pr-S-
b
GSH
Control
Ang II
10–7 M
GSS-Ras
b GS-SPr
Ras
SA Pull-down
Blot p21ras
Control
-7
-8
-9
H2O2
AII log [mol/L] (250 M)
(15 min)
Adachi, JBC 2004
Increase in intracellular GSH caused by
biotinylated-GSH ester in BAEC
Fold Increase
4
**
3
2
1
0
Control
BGSH
BGSH SA
Clavreul et al. FASEB J, 2006
S-Glutathiolated proteins detected with
35S-GSH
[35S] cysteine
Cycloheximide
Fratelli, PNAS
2002
IP
2D-Gel
S-Glutathiolation of p21ras in Endothelial Cells
Exposed to Oxidized LDL: Anti-GSH Antibody
DTT
oxLDL
-
+
IP: p21ras
p21ras-IB: GSH
+
+
--21 kDa
GSS-p21ras
Fold Increase
4
+
-
*
3
2
1
0
LacZ
LacZ C118S Grx C118S Grx
oxLDL
Clavreul et al. ATVB 2006
1
2
3
118-CDLAAR-123
953.43
IP
p21ras
953.32
S-Glutathiolated p21ras in Endothelial Cells
Exposed to Oxidized LDL: MALDI-TOF
SSG
100
75
50
37
OxLDL
OxLDL
+ DTT
1397.63
20
1397.69
150-QGVEDAFYTLVR-161
25
15
-
Coomassie Anti -GSH
IB
GSH
Clavreul et al. ATVB, 2006
Acknowledgements:
Vascular Biology Unit:
Nicolas Clavreul
Ardelle Jia Ying
Takeshi Adachi
Reiko Matsui
Maha Sethuraman
Bingbing Jiang
Mengwei Zang
Xiaoyong Tong
Markus Bachschmid
Stefan Schildknecht
Tyler Heibeck
Hossein Bayat
Rebecca Zee
Robert Weisbrod
Shanqin Xu
Karlene Maitland
Chaomei Shi
Xuiyun Hou
Cardiovascular Proteomics Center
Mark McComb
Claire Dauly
Peter O’Connor
Cheng Zhao
Cathy Costello
University of Kansas
Christian Schoeneich
Viktor Sharov