proteomics of protein glutathionylation in mitochondria of an aging heart jessica page mentor: dr....
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Proteomics of Protein Proteomics of Protein Glutathionylation in Glutathionylation in Mitochondria of an Mitochondria of an
Aging HeartAging Heart
Jessica PageJessica Page
Mentor: Dr. Claudia MaierMentor: Dr. Claudia Maier
Department of ChemistryDepartment of Chemistry
Mitochondria
Protein GlutathionylationProtein Glutathionylation
ROS create an ROS create an imbalance between imbalance between glutathione (GSH) glutathione (GSH) and glutathione and glutathione disulfide (GSSH)disulfide (GSSH)
Fig. 1. Structure of glutathione (GSH) , a tripeptide comprised of three amino acids, glutamic acid, cysteine and glycine.
Fig. 1. Structure of glutathione (GSH) , a tripeptide comprised of three amino acids, glutamic acid, cysteine and glycine.
Fig. 1. Structure of glutathione (GSH) , a tripeptide comprised of three amino acids, glutamic acid, cysteine and glycine.
Glutathionylation: Glutathionylation: Modification of proteins Modification of proteins with GSSGwith GSSG
Protein S SGATCICys- SH
Protein S S Cys-ICAT GSH
+
Protein-SH + GSSG Protein-S-SG + GSH Glutathionylation
2 GSH GSSG
GoalGoal
Does damage to mitochondria as a result of protein oxidation and glutathionylation contribute to the loss of mitochondrial
function in the aging heart?
Specific AimsSpecific Aims
Personal aimsPersonal aims
Biotin
Linker Functional Group
Affinity Attachment
Affinity Chromatograph
y
Gel Electrophoresis
Western Blot
Streptavidin
Probe Components
and Functionaliti
es
Probe Synthesis
NH2
SS
NH2
O
O
O O
cystine
NH
SS
NH
O
O
O O
NH
NH
O H
NH
HS
O O O
NH
O
O
NH
NH
OH
NH
HS
OOO
NH
O
O
NH
NH
O H
NH
HS
O O O
NH
OOH
O+
PBSA
Cys-ICAT dimer
1170 1180 1190 m/z
0
100
%
1181.5272
1182.5375
1183.5480
1184.5430
Cys-ICAT (dimer)
1180.527JMP004
MH+
NH
SS
NH
O
O
O O
NH
NH
O H
NH
HS
O O O
NH
O
O
NH
NH
OH
NH
HS
OOO
NH
O
O
NH
SS
NH
O
O
O O
NH
NH
O H
NH
HS
O O O
NH
O
O
NH
NH
OH
NH
HS
OOO
NH
O
O
NH
NHO
S
H
H
NH
O
OO
NH
O
NH
O
SHO
O
Cys-ICAT
Cys-ICAT dimer
reduction
580 585 590 595 600 605 610m/z0
100
%
592.2659
593.2763
594.2650
595.5048
MH+
Cys-ICAT591.2659JMP004
Reduction using TCEP
NH
NHO
S
H
H
NH
O
OO
NH
O
NH
O
SO
O
Protein
S
NH NH
OH
O
O
O
O
Protein
NH2NH
NHO
S
H
H
NH
O
OO
NH
O
NH
O
SHO
O
Cys-ICAT
+
Title
Proof of Concept: Probe + GSSG
SDS-Page/Blot/Spot
899 1159 1419 1679 1939 2200
Mass (m/z)
4890
0
10
20
30
40
50
60
70
80
90
100
% In
ten
sity
1777.0
1102.6
976.6
1648.9
1913.0 2109.1
2041.0
Survey Scan Of Spot 22
69 395 721 1047 1373 1700
Mass (m/z)
1479
0
10
20
30
40
50
60
70
80
90
100
% Intensity
303y2
507y4
641b6
732y6
738b7
301b3
1136y10
1235y11
1591
1476
y13
1039y9
360y3
1630b15
y14
G Q D I K V P P P L P Q F G R Kb3 b6
y2y3y4y6
b7
y9y10y11y13
b15
y14
MS/MS 1777.0 (1+)
69 395 721 1047 1373 1700
Mass (m/z)
1479
0
10
20
30
40
50
60
70
80
90
100
% Intensity
303y2
507y4
641b6
732y6
738b7
301b3
1136y10
1235y11
1591
1476
y13
1039y9
360y3
1630b15
y14
G Q D I K V P P P L P Q F G R Kb3 b6
y2y3y4y6
b7
y9y10y11y13
b15
y14
G Q D I K V P P P L P Q F G R Kb3 b6
y2y3y4y6
b7
y9y10y11y13
b15
y14
MS/MS 1777.0 (1+)
22 23
3017 24
28
27
33 26
25
1514
3534
12111819
313221
12
22 23
3017 24
28
27
33 26
25
1514
3534
12111819
313221
12
Step 1: Protein Separation: 2D Gel Step 2: Excise Gel SpotStep 3: Proteolyze/extract peptidesStep 4: Mass Spectrometry
Peptide Mass Fingerprint
Sequence Tag
Step 5: Protein Identity(Database Search)
Future: Combination of MS-Based Proteomic and MY probe
Creatine Kinase