Extending Microarray Technology to Study Protein Function...
Transcript of Extending Microarray Technology to Study Protein Function...
Extending Microarray Technologyto Study Protein Function
Gavin MacBeathBauer Center for Genomics Research
Harvard University
MICROARRAYS: THE POWER OF MULTIPLEXING
Samples in 96-or 384-well plates
Slit width:~ 30 µmarrayer
print head
split pins
simultaneous analysis
cDNA microarrays
• quantitate relative abundance of >10,000
different mRNAs between two cell samples
repeated analysis
cDNA microarrays
• quantitate >10,000 mRNAs from cells under
>100 different conditions
• cluster genes that are co-regulated
MICROARRAYS: THE POWER OF MULTIPLEXING
Samples in 96-or 384-well plates
Slit width:~ 30 µmarrayer
print head
split pins
protein microarrays
• screen >1000 proteins simultaneously
simultaneous analysis repeated analysis
protein microarrays
• screen >1000 proteins with >1000 proteins
• screen >100 protein-protein interactions
with >10,000 small molecules (competition assay)
KEEP THE PROTEINS HAPPY
• hydrophilic surface
• amine-reactive chemistry
• constant hydration
OH
OH
OHEtO Si
OEtOEt
H2N (1)
Glass Slide
(2)
NO O
NO
O
O
O
O
(4)
NO O
NO
O
O
O
O
BSA-NHS Slide
(3)
BSA
O
O
O Si
Si
Si
O
O
OH
OH HN
NH2
HN
O
O
HN
HN
O
O
N
O
O
HN O
O
N
O
OBSA
O
O
O Si
Si
Si
O
O
OH
OH HN
NH2
HN
O
O
HN
HN
O
HN
O
BSA
HN
HN CO2
-H2N
H2N CO2-(2)
(1)
A
B
BSA-NHS SLIDES
OH
OH
OH
Glass Slide Aldehyde Slide
EtO Si OEtOEt
O
H
H2N
O SiO
SiO
OH
O
H
O
H
OSiOOH
O
H
N
O SiO
SiO
OH
N
H
H
OSiOOH
H BSAN
H2N
BSAH2N
(1)
(2)
A
B
ALDEHYDE SLIDES
PROTEIN MICROARRAYS
protein-protein
interactions
enzyme-substrate
interactions
protein-small molecule
interactions
protein-protein
interactions
Glass Slide
Fluorophore
PROTEIN-PROTEIN INTERACTIONS
p50
GST-FRB
0.50 µg/ml BODIPY-FL-IgG0.05 µg/ml Cy3-IΚBα0.50 µg/ml Cy5-FKBP12
Protein G
1 mm
100 nM rapamycin: - +
PROTEIN-PROTEIN INTERACTIONS
+1 cm
Column 109
Row
27
0.50 µg/ml BODIPY-FL-IgG0.50 µg/ml Cy5-FKBP12 + 100 nM rapamycin
PROTEIN-PROTEIN INTERACTIONS
TITRATION ON GLASS SLIDE
100
1,000
10,000
100,000
1,000,000
10,000,000
0.0001 0.001 0.01 0.1 1 10
[Cy5-FKBP12] (µg/ml)
Fluore
scence
units
GST-FRB2000 µg/ml1000 µg/ml500 µg/ml
150 pg/ml (12.5 pM)
PROTEIN MICROARRAYS
protein-protein
interactions
enzyme-substrate
interactions
protein-small molecule
interactions
enzyme-substrate
interactions
Radioactive
ATP ADP
Kinase
P
Glass Slide
ENZYME-SUBSTRATE INTERACTIONS IDENTIFYING SUBSTRATES OF PROTEIN KINASES
I-2
Elk1
Kemptide
1 mm
PKA CK II Erk2
PROTEIN MICROARRAYS
protein-protein
interactions
enzyme-substrate
interactions
protein-small molecule
interactions
protein-small molecule
interactions
FluorophoreBSA
SmallMolecule
BSA
Glass Slide
PROTEIN-SMALL MOLECULE INTERACTIONS
IDENTIFYING THE TARGETS OF SMALL MOLECULES
Streptavidin
(His)6-FKBP12
10 µg/ml A488-BSA-digoxigenin
5 µg/ml Cy3-BSA-AP1497
5 µg/ml Cy5-BSA-biotin
Anti-Dig-IgG
1 mm
PROTEIN MICROARRAYS
functionalgenomics
proteinprofiling
small moleculediscovery
subsets of related proteins
protein domains full-length proteins
• informatics (sequence motifs)• literature searches
• coiled coilsJohn Newman
• protocadherinsViara Grantcharova
• cancer proteinsJoshua LaBaer, Pascal Braun
• c. elegans developmentViara Grantcharova
FUNCTIONAL GENOMICS
Mediate homo-and hetero-dimerization
Found in:• structural proteins• motor proteins• transcription factors• membrane fusion proteins
In yeast, MULTICOIL predicts:• ~300 proteins with
2-stranded coiled-coils• ~250 proteins with
3-stranded coiled coils
COILED COILS
tropomyosinhomodimer
myc/maxheterodimer
J Mol Biol (1998) 281, 165. J Mol Biol (1986) 192, 111.
COILED-COIL SCREEN: YEAST TWO-HYBRID
Pilot Screen
Newman, Wolf & Kim (2000) PNAS 97, 13203
DNA Binding Domain Constructs
Act
ivat
ion
Dom
ain
Const
ruct
s
20 homotypic
19 heterotypic
3 intra-protein
1
68
1 68
Cy3-Gcn4p
Cy3-Met4p
Cy3-Met28p
Cy3-Spc42p
Cy3-Myc
Cy3-Mxi1
Cy3-Mad
Cy3-Max
COILED-COIL INTERACTIONS: PROTEIN MICROARRAYS
-
Met28pGcn4p Met4p Spc42p
MaxMadMyc Mxi1
John Newman
Prote
ins
1-7
2
Cy3-Met28p / Met4p
Cy3-Spc42p / Spc42p
PILOT COILED-COIL SCREEN: PROTEIN MICROARRAYS
John Newman
functionalgenomics
proteinprofiling
PROTEIN MICROARRAYS
small moleculediscovery
SCREENING METHOD 2
protein microarrays
SCREENING METHOD 2
wash and scan
protein microarrays
SCREENING METHOD 2
protein microarrays
SCREENING METHOD 2
protein microarrays
small moleculelibrary
each well containsall the labeled ligandsand a different small molecule
SCREENING METHOD 2
protein microarrays
SCREENING METHOD 2
protein microarrays
wash and scan
THE MICROARRAY WORLD
Glass slides
• 2.5 cm x 7.5 cm• >10,000 spots per plate• spacing varies
THE HTS WORLD
Microtiter plates
• 8.5 cm x 12.5 cm• 96, 384, or 1536 wells per plate• 9 mm, 4.5 mm, or 2.25 mm spacing
MICROARRAYS IN WELLS OF PLATES
silicone gasket
bottomless384-well plate
protein arrayson glass slides
strong adhesive
weaker,reversible,
water-tight seal
MULTIPLEXED SCREENING
• bottomless 384-well plate• 64 wells per slide x 4 slides per plate = 256 wells per plate• 256 wells per plate x 100 proteins per well = 25,600 assays per plate
proteinprofiling
functionalgenomics
PROTEIN MICROARRAYS
small moleculediscovery
ANTIBODY ARRAYS: SANDWICH ELISA
untreatedcells
lyse lyse
fractionate fractionate
treatedcells
1. apply unlabeled protein
2. detect with labeledsecondary antibody
Antigen
+ TfR + ErbB2 + EGFR
+ TfR+ ErbB2+ EGFR
anti-TfR capture mAb
anti-ErbB2 capture mAb
anti-EGFR capture mAb
detected with mixture of: anti-TfR mAb#2-CY3 anti-ErbB2 mAb#2-Alexa488 anti-EGFR mAb#2-CY5
ANTIBODY ARRAYS: MICRO-SANDWICH ASSAY
Flourescent 2' Antibodies
Capture Antibody
TfR
ErbB2
EGFR
0
1000
3000
2000
TfR
ErbB2
EGFR
0
1000
3000
2000
TfR
ErbB2
EGFR
0
1000
3000
2000
TfR
ErbB2
EGFR
0
1000
3000
2000
0
250
500
0
250
500
0
250
500
0
250
500
A-431squamouscarcinoma
SK-BR-3breast
carcinoma
MCF-7transfected
ErbB2
MCF-7breast
carcinoma
micro-arrayflow
cytometry
MICRO-SANDWICH DETECTIONTUMOR CELL PROFILING
glass slide
ANTIBODY ARRAYS: MICRO-SANDWICH ASSAY
antigen
PO4PO4 PO4
fluorophore
Ratiometric analyses of protein modification
- EGFR is phosphorylated in response to EGF- ErbB2 is phosphorylated by EGFR- EGFR is down-regulated- TfR remains unchanged
EGFREGFR
EGFR
EGFR
Erb
B2TfR
EGF
PHOSPHORYLATION AND REGULATION OF GROWTH FACTOR RECEPTORS
PO4 PO
4
RATIOMETRIC ANALYSES OF EXPRESSION AND PHOSPHORYLATION
Blue - Alexa488; Green - Cy3; Red - Cy5Capture and detection antibodies against different epitopes
InterrogateTfR expression
(internal reference)
TfR
α-TfR
α-TfR
InterrogateErbB2 expression +
Y-1248 phosphorylation
ErbB24
α-ErbB2
PO4
α-ErbB2α-pY1248
InterrogateEGFR expression +
Y-1068 phosphorylation
EGFRPO4
α-EGFR
PO4
α-EGFRα-pY1068
EFFECT OF EGF
RATIOMETRIC PROFILING IN TUMOR CELL LINES
RED = growth factor receptor expressionGREEN = growth factor receptor phosphorylationBLUE = transferrin receptor expressionYELLOW = ratio expression/phosphorylation
anti-EGFR
anit-ErbB2
anti-TfR
EGF
A-431squamouscarcinoma
+ -
SK-BR-3breast
carcinoma
+ -
MCF-7breast
carcinoma
+ -
MCF-7transfected
ErbB2
+ -
EGFR
EGFR
Erb
B2TfR
EGF
PO4 PO
4
RATIOMETRIC ANALYSES OF EXPRESSION AND PHOSPHORYLATION
EGF time course on SK-BR-3 cells
TfR
EGFREGFR
EGFR
EGFR
ErbB2
EGF
PO4 PO
4
time of EGF treatment
anti-EGFRanit-ErbB2
anti-TfR
0 m
in
1 m
in
5 m
in
15 m
in
60 m
in
120
min
0 min
1 min
5 min
15 m
in
60 m
in
120 m
in
0
100
200
300
400
500
anti-EGFRY1068
0
300
600
900
1200
anti-EGFR
0 min
1 min
5 min
15 m
in
60 m
in
120 m
in 0
0.1
0.2
0.3
0.4
0.5
0 min
1 min
5 min
15 m
in
60 m
in
120 m
in
PO4-Y1068/EGFR
anti-ErbB2Y1248
0
250
500
750
1000
0 min
1 min
5 min
15 m
in
60 m
in
120 m
in
0
1000
2000
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anti-ErbB2
0 min
1 min
5 min
15 m
in
60 m
in
120 m
in
PO4-Y1068/ErbB2
0
0.1
0.2
0.3
0.4
0 min
1 min
5 min
15 m
in
60 m
in
120 m
inRATIOMETRIC ANALYSES OF EXPRESSION AND PHOSPHORYLATION
EGF time course on SK-BR-3 cells
EGFR/pY1068
ErbB2/pY1248
phoshorylationratio
anti-TfR
0
1000
2000
3000
0 min
1 min
5 min
15 m
in
60 m
in
120 m
in
TfR
receptorexpression
receptorphosphorylation functional
genomicsproteinprofiling
• rapid discovery of protein interactions• screening of small molecules in solution• screening for specificity• system-wide analysis at the protein level
SUMMARY
PROTEIN MICROARRAYS
small moleculediscovery
ACKNOWLEDGEMENTS
Antibody Microarrays
Prof. Peter Sorger (MIT)Dr. Ulrik Nielsen (MIT)Dr. Michael Cardone (MIT)
Dr. Raghida Bu-Khalid
Harvard Center for Genomics ResearchDARPA
Protein Microarrays
Prof. Peter Kim (Whitehead Institute)Dr. John Newman (Whitehead Institute)
Dr. Viara GrantcharovaLioudmila Zaslavskaia