MICROARRAYS: THE POWER OF MULTIPLEXING Extending Microarray Technology print head to Study Protein Function Samples in 96- or 384-well plates arrayer Slit width: ~ 30 µm Gavin MacBeath split pins Bauer Center for Genomics Research Harvard University simultaneous analysis repeated analysis cDNA microarrays cDNA microarrays • quantitate >10,000 mRNAs from cells under • quantitate relative abundance of >10,000 >100 different conditions different mRNAs between two cell samples • cluster genes that are co-regulated MICROARRAYS: THE POWER OF MULTIPLEXING KEEP THE PROTEINS HAPPY print head • hydrophilic surface Samples in 96- or 384-well plates • amine-reactive chemistry arrayer Slit width: ~ 30 µm split pins • constant hydration simultaneous analysis repeated analysis protein microarrays protein microarrays • screen >1000 proteins with >1000 proteins • screen >1000 proteins simultaneously • screen >100 protein-protein interactions with >10,000 small molecules (competition assay)
BSA-NHS SLIDES ALDEHYDE SLIDES H 2 N (1) (2) O O O OH H N O O N O OH O Si H O O EtO Si OEt OH N A O H H O H OH OEt O O O Si N N OH O O Si O O BSA O EtO Si OEt O O H A OH O Si NH 2 OH OEt O Si O O O H H H OH N O OH O Si N N N (3) (4) OH O O O O N O O N O BSA Aldehyde Slide Glass Slide O Glass Slide BSA-NHS Slide O O (1) H OH H - H 2 N N CO 2 (1) OH H 2 N O Si H H N O Si N N O B O H BSA O O Si O B O Si NH 2 N O H BSA O H H H H O Si N N (2) N O Si N N H 2 N BSA OH (2) - H 2 N CO 2 OH O O H 2 N PROTEIN MICROARRAYS PROTEIN-PROTEIN INTERACTIONS Fluorophore protein- protein- enzyme- protein- Glass Slide protein protein substrate small molecule interactions interactions interactions interactions
PROTEIN-PROTEIN INTERACTIONS PROTEIN-PROTEIN INTERACTIONS Column 109 Row 27 Protein G 1 mm p50 GST-FRB 1 cm 100 nM rapamycin: - + + 0.50 µg/ml BODIPY-FL-IgG 0.50 µg/ml BODIPY-FL-IgG 0.50 µg/ml Cy5-FKBP12 + 100 nM rapamycin 0.05 µg/ml Cy3-I Κ B α 0.50 µg/ml Cy5-FKBP12 TITRATION ON GLASS SLIDE PROTEIN MICROARRAYS 10,000,000 2000 µ g/ml GST-FRB 1000 µ g/ml 500 µ g/ml 1,000,000 Fluorescence units 100,000 10,000 protein- enzyme- enzyme- protein- 1,000 150 pg/ml (12.5 pM) protein substrate substrate small molecule interactions interactions interactions interactions 100 0.0001 0.001 0.01 0.1 1 10 [Cy5-FKBP12] ( µ g/ml)
ENZYME-SUBSTRATE INTERACTIONS IDENTIFYING SUBSTRATES OF PROTEIN KINASES Kemptide Kinase Radioactive ADP ATP 1 mm I-2 P Elk1 Glass Slide PKA CK II Erk2 PROTEIN MICROARRAYS PROTEIN-SMALL MOLECULE INTERACTIONS Fluorophore BSA Small Molecule BSA protein- enzyme- protein- protein- protein substrate small molecule small molecule Glass Slide interactions interactions interactions interactions
IDENTIFYING THE TARGETS OF SMALL MOLECULES PROTEIN MICROARRAYS Anti-Dig-IgG functional small molecule protein genomics discovery profiling Streptavidin 1 mm (His) 6 -FKBP12 10 µg/ml A 488 -BSA-digoxigenin 5 µg/ml Cy3-BSA-AP1497 5 µg/ml Cy5-BSA-biotin FUNCTIONAL GENOMICS COILED COILS subsets of related proteins Mediate homo- and hetero-dimerization • informatics (sequence motifs) Found in: • literature searches • structural proteins • motor proteins • transcription factors • membrane fusion proteins In yeast, MULTICOIL predicts: protein domains full-length proteins • ~300 proteins with 2-stranded coiled-coils • coiled coils • cancer proteins • ~250 proteins with 3-stranded coiled coils John Newman Joshua LaBaer, Pascal Braun • protocadherins • c . elegans development myc/max tropomyosin Viara Grantcharova Viara Grantcharova heterodimer homodimer J Mol Biol (1998) 281 , 165. J Mol Biol (1986) 192 , 111.
COILED-COIL SCREEN: YEAST TWO-HYBRID COILED-COIL INTERACTIONS: PROTEIN MICROARRAYS Pilot Screen Cy3-Gcn4p Cy3-Myc DNA Binding Domain Constructs 1 68 1 Cy3-Met4p Cy3-Mxi1 Activation Domain Constructs Cy3-Met28p Cy3-Mad 20 homotypic 19 heterotypic Cy3-Spc42p Cy3-Max 3 intra-protein Gcn4p Met4p Met28p Spc42p 68 Myc Mxi1 Mad - Max Newman, Wolf & Kim (2000) PNAS 97 , 13203 John Newman PILOT COILED-COIL SCREEN: PROTEIN MICROARRAYS PROTEIN MICROARRAYS functional small molecule protein Proteins 1-72 genomics discovery profiling Cy3-Spc42p / Spc42p Cy3-Met28p / Met4p John Newman
SCREENING METHOD 2 SCREENING METHOD 2 wash and scan protein microarrays protein microarrays SCREENING METHOD 2 SCREENING METHOD 2 small molecule library each well contains all the labeled ligands and a different small molecule protein microarrays protein microarrays
SCREENING METHOD 2 SCREENING METHOD 2 wash and scan protein microarrays protein microarrays THE MICROARRAY WORLD THE HTS WORLD MICROARRAYS IN WELLS OF PLATES bottomless 384-well plate strong adhesive silicone gasket Glass slides Microtiter plates • 2.5 cm x 7.5 cm • 8.5 cm x 12.5 cm weaker, • >10,000 spots per plate • 96, 384, or 1536 wells per plate reversible, • spacing varies • 9 mm, 4.5 mm, or 2.25 mm spacing water-tight seal protein arrays on glass slides
MULTIPLEXED SCREENING PROTEIN MICROARRAYS functional small molecule protein genomics discovery profiling • 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 ANTIBODY ARRAYS: SANDWICH ELISA ANTIBODY ARRAYS: MICRO-SANDWICH ASSAY Flourescent 2' Antibodies untreated treated cells cells 1. apply unlabeled protein 2. detect with labeled secondary antibody Antigen Capture Antibody lyse lyse + TfR + ErbB2 + TfR + ErbB2 + EGFR + EGFR fractionate fractionate 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
MICRO-SANDWICH DETECTION TUMOR CELL PROFILING flow micro-array cytometry ANTIBODY ARRAYS: MICRO-SANDWICH ASSAY A-431 TfR squamous ErbB2 Ratiometric analyses of protein modification carcinoma EGFR 0 1000 2000 3000 250 500 0 fluorophore SK-BR-3 TfR breast ErbB2 carcinoma PO EGFR PO PO 4 4 4 0 1000 2000 3000 0 250 500 antigen MCF-7 TfR breast ErbB2 EGFR carcinoma glass slide 1000 2000 3000 250 500 0 0 MCF-7 TfR transfected ErbB2 ErbB2 EGFR 0 1000 2000 3000 0 250 500 PHOSPHORYLATION AND REGULATION OF RATIOMETRIC ANALYSES OF GROWTH FACTOR RECEPTORS EXPRESSION AND PHOSPHORYLATION EGF Interrogate Interrogate Interrogate EGFR expression + ErbB2 expression + TfR expression EGFR EGFR Y-1068 phosphorylation Y-1248 phosphorylation (internal reference) ErbB2 TfR α -pY1068 α -pY1248 α -EGFR α -ErbB2 P O α -TfR PO 4 4 PO PO PO 4 EGFR 4 4 4 EGFR TfR EGFR ErbB2 α -EGFR α -ErbB2 α -TfR - EGFR is phosphorylated in response to EGF Capture and detection antibodies against different epitopes - ErbB2 is phosphorylated by EGFR Blue - Alexa488; Green - Cy3; Red - Cy5 - EGFR is down-regulated - TfR remains unchanged
RATIOMETRIC PROFILING RATIOMETRIC ANALYSES OF IN TUMOR CELL LINES EXPRESSION AND PHOSPHORYLATION EFFECT OF EGF EGF time course on SK-BR-3 cells EGF EGF EGFR EGFR EGFR EGFR ErbB2 ErbB2 TfR TfR P O PO P O 4 PO 4 4 4 EGFR EGFR A-431 SK-BR-3 MCF-7 MCF-7 squamous breast breast transfected carcinoma carcinoma carcinoma ErbB2 anti-TfR anit-ErbB2 anti-EGFR - + - + - + - + anti-TfR EGF anit-ErbB2 anti-EGFR RED = growth factor receptor expression 0 min 1 min 5 min 15 min 60 min 120 min GREEN = growth factor receptor phosphorylation BLUE = transferrin receptor expression YELLOW = ratio expression/phosphorylation time of EGF treatment RATIOMETRIC ANALYSES OF EXPRESSION AND PHOSPHORYLATION PROTEIN MICROARRAYS EGF time course on SK-BR-3 cells receptor receptor phoshorylation expression phosphorylation ratio functional small molecule protein genomics discovery profiling anti-EGFR PO4-Y1068/ anti-EGFR Y1068 EGFR 1200 500 0.5 EGFR/ 400 0.4 900 300 0.3 pY1068 600 200 0.2 300 100 0.1 0 0 0 0 min 1 min 5 min 15 min 60 min 120 min 0 min 1 min 5 min 15 min 60 min 120 min 0 min 1 min 5 min 15 min 60 min 120 min anti-ErbB2 PO4-Y1068/ anti-ErbB2 Y1248 ErbB2 4000 0.4 ErbB2/ 1000 3000 750 0.3 pY1248 2000 500 0.2 SUMMARY 1000 0.1 250 0 0 0 0 min 1 min 5 min 15 min 60 min 120 min • rapid discovery of protein interactions 0 min 1 min 5 min 15 min 60 min 120 min 0 min 1 min 5 min 15 min 60 min 120 min • screening of small molecules in solution anti-TfR • screening for specificity 3000 • system-wide analysis at the protein level TfR 2000 1000 0 0 min 1 min 5 min 15 min 60 min 120 min
ACKNOWLEDGEMENTS Protein Microarrays Antibody Microarrays Prof. Peter Kim (Whitehead Institute) Prof. Peter Sorger (MIT) Dr. John Newman (Whitehead Institute) Dr. Ulrik Nielsen (MIT) Dr. Michael Cardone (MIT) Dr. Viara Grantcharova Lioudmila Zaslavskaia Dr. Raghida Bu-Khalid Harvard Center for Genomics Research DARPA
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