A biochemists' tool kit to study the battlefront up close (the techniques used to study...
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A biochemists' tool kit to study the battlefront up close
(the techniques used to study protein--protein interactions)
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ProteomicsProteome = All the proteins expressed
by a specific genome or tissue
gtc tac ctc cag acc tcc ttg aaa tac aat att ctc cca gaa aag gaa gag
Gene sequence (DNA)
Protein sequenceK C R V N C F K L S I L P E K E E
But, genes can be translated several ways to create >1 protein per geneAnd then, proteins can be modified to create >1 form of the protein per sequence
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How a MALDI mass spectrometer works
m/z
Ion counts
1000 2000 3000
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Data System
Mass spectrum
out
Inlet System
Mass Analyzer
Ion Source
Detector
Vacuum envelope
Sample in
Inlet systems: •Simple vacuum lock•HPLC•GC
Ion sources:
Mass analyzers:
•Electrospray (ESI)•MALDI•FAB/LSIMS•Electron ionization (EI)
•Quadrupole•Time-of-flight•Ion trap•Magnetic sector•FTMS
Mass spectrometer schematics
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Targeted Proteomics
Fish with “bait”(interesting protein)
Pull-out interacting proteins
Excise bands,Identify proteins
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Cellular Proteomics
Cell in state A Cell in state B
Digest all proteins, LC-MS/MS
Compare 2D gels
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The human interactome (500 baits)
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LRP1 may provide the connection between damage inside and outside neurons in Alzheimer’s
TNFT
NPVYA
TLY
TNFTNPV
pYATLY
PI3 kinase p85
PLCγ-1
Histone Deacetylase 6
PI3 kinase p110 & GAP1
SYP
Sos-1
CRAF1, Shp1, Shp2
CaM kinase II Src
CSK
Elongation Factor 1α Crkl
Ck1-αPyrroline – 5 carboxylate reductase
Ribosomal S4
Grb2
Shc
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Heart disease involves too much blood clotting
Thrombin + Thrombomodulin
antithrombinIII
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Blood Vessel Endothelium
Thrombin
TM
Protein C
ActivatedProtein C
Thrombin-TM
The thrombin-thrombomodulin interaction takes place in the flowing blood
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05
101520
0 50 100 150 200 250 300
Thrombin
TM
Thrombin-TM
q
Thrombin Injection Buffer Injection
Time (in seconds)
Surface plasmon resonance: real-time binding kinetics of interactions in a flowing system
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0
5
10
15
20
25
30
35
time (s)
(B)
0
5
10
15
20
25
30
35
0 40 80 120160200time (s)
(A)
0 100 200 300 400
Thrombin-mAbThrombin-TM456
The thrombin-thrombomodulin interaction is fast-on, fast-off
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DD DDD DD
DH2O
DD HH
D DH
H H
pepsinD
DD
DH
H
The off-exchange experiment measures retention of amide deuteration at protein-protein interfaces.
The on-exchange experiment measures solvent accessibility
H DDH HH D DD
t (short) t (long)DH
H H
pepsinD2OD2O
The MALDI-Mapping Experiments
Both on-exchange and off-exchange data are required to distinguish interface protection from conformational changes.
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MALDI-Mapping shows that Thrombomodulin changes the active site of thrombin
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Conclusions
• Mass spectrometry is useful for finding protein-protein interaction binding partners
• Our lab is finding new target sites for Alzheimer’s Disease
• MALDI-mapping helps find binding sites
• Our lab is using MALDI mapping to find activity changes in proteins induced by an interaction partner