A biochemists' tool kit to study the battlefront up close

(the techniques used to study protein--protein interactions)

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

How a MALDI mass spectrometer works

m/z

Ion counts

1000 2000 3000

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

Targeted Proteomics

Fish with “bait”(interesting protein)

Pull-out interacting proteins

Excise bands,Identify proteins

Cellular Proteomics

Cell in state A Cell in state B

Digest all proteins, LC-MS/MS

Compare 2D gels

The human interactome (500 baits)

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

Heart disease involves too much blood clotting

Thrombin + Thrombomodulin

antithrombinIII

Blood Vessel Endothelium

Thrombin

TM

Protein C

ActivatedProtein C

Thrombin-TM

The thrombin-thrombomodulin interaction takes place in the flowing blood

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

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

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.

MALDI-Mapping shows that Thrombomodulin changes the active site of thrombin

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