Extending Microarray Technology to Study Protein Function

Transcription

1 MICRARRAYS: TE PWER F MULTIPLEXING Extending Microarray Technology to Study Protein Function Samples in 96- or 384-well plates print head Gavin MacBeath Bauer Center for Genomics Research arvard University arrayer Slit width: ~3µm split pins simultaneous analysis cdna microarrays quantitate relative abundance of >1, different mrnas between two cell samples repeated analysis cdna microarrays quantitate >1, mrnas from cells under >1 different conditions cluster genes that are co-regulated MICRARRAYS: TE PWER F MULTIPLEXING KEEP TE PRTEINS APPY print head hydrophilic surface Samples in 96- or 384-well plates arrayer Slit width: ~3µm amine-reactive chemistry split pins constant hydration simultaneous analysis repeated analysis screen >1 proteins simultaneously screen >1 proteins with >1 proteins screen >1 protein-protein with >1, small molecules (competition assay)

2 -NS SLIDES ALDEYDE SLIDES A Glass Slide 2 N (1) Et Si Et Et (3) (2) N N (4) N N Si Si Si N N N 2 N N -NS Slide N N N A Glass Slide Et Si Et Et Si Si Si Aldehyde Slide B (1) (2) 2 N 2 N C 2 - Si Si Si N N N 2 N N N C2 - N N B (1) (2) 2 N 2 N 2 N Si Si Si N N N PRTEIN MICRARRAYS PRTEIN-PRTEIN INTERACTINS Fluorophore proteinprotein enzymesubstrate proteinsmall molecule Glass Slide

3 PRTEIN-PRTEIN INTERACTINS PRTEIN-PRTEIN INTERACTINS Column 19 Protein G Row 27 p5 1mm GST-FRB 1 nm rapamycin: µg/ml BDIPY-FL-IgG.5 µg/ml Cy3-IΚBα.5 µg/ml Cy5-FKBP12 1cm.5 µg/ml BDIPY-FL-IgG.5 µg/ml Cy5-FKBP nm rapamycin TITRATIN N GLASS SLIDE PRTEIN MICRARRAYS 1,, 1,, 2 µg/ml 1 µg/ml 5 µg/ml GST-FRB Fluorescence units 1, 1, 1, 15 pg/ml (12.5 pm) proteinprotein enzymesubstrate proteinsmall molecule [Cy5-FKBP12] (µg/ml)

4 ENZYME-SUBSTRATE INTERACTINS IDENTIFYING SUBSTRATES F PRTEIN KINASES Kinase Radioactive Kemptide ATP ADP P I-2 1mm Glass Slide Elk1 PKA CK II Erk2 PRTEIN MICRARRAYS PRTEIN-SMALL MLECULE INTERACTINS Fluorophore Small Molecule proteinprotein enzymesubstrate proteinsmall molecule Glass Slide

5 IDENTIFYING TE TARGETS F SMALL MLECULES PRTEIN MICRARRAYS Anti-Dig-IgG Streptavidin 1mm functional genomics small molecule discovery protein profiling (is) 6 -FKBP12 1 µg/ml A digoxigenin 5 µg/ml Cy3--AP µg/ml Cy5--biotin FUNCTINAL GENMICS CILED CILS subsets of related proteins informatics (sequence motifs) literature searches Mediate homoand hetero-dimerization Found in: structural proteins motor proteins transcription factors membrane fusion proteins protein domains coiled coils John Newman full-length proteins cancer proteins Joshua LaBaer, Pascal Braun In yeast, MULTICIL predicts: ~3 proteins with 2-stranded coiled-coils ~25 proteins with 3-stranded coiled coils protocadherins Viara Grantcharova c. elegans development Viara Grantcharova myc/max heterodimer tropomyosin homodimer J Mol Biol (1998) 281, 165. J Mol Biol (1986) 192, 111.

6 CILED-CIL SCREEN: YEAST TW-YBRID CILED-CIL INTERACTINS: PRTEIN MICRARRAYS Pilot Screen Cy3-Gcn4p Cy3-Myc DNA Binding Domain Constructs Cy3-Met4p Cy3-Mxi1 Activation Domain Constructs 2 homotypic 19 heterotypic 3 intra-protein Cy3-Met28p Cy3-Spc42p Cy3-Mad Cy3-Max 68 Gcn4p Met4p Met28p Spc42p Myc Mxi1 Mad - Max Newman, Wolf & Kim (2) PNAS 97, 1323 John Newman PILT CILED-CIL SCREEN: PRTEIN MICRARRAYS PRTEIN MICRARRAYS Proteins 1-72 Cy3-Spc42p / Spc42p functional genomics small molecule discovery protein profiling Cy3-Met28p / Met4p John Newman

7 SCREENING METD 2 SCREENING METD 2 wash and scan SCREENING METD 2 SCREENING METD 2 small molecule library each well contains all the labeled ligands and a different small molecule

8 SCREENING METD 2 SCREENING METD 2 wash and scan TE MICRARRAY WRLD TE TS WRLD MICRARRAYS IN WELLS F PLATES bottomless 384-well plate strong adhesive Glass slides Microtiter plates silicone gasket 2.5 cm x 7.5 cm >1, spots per plate spacing varies 8.5 cm x 12.5 cm 96, 384, or 1536 wells per plate 9 mm, 4.5 mm, or 2.25 mm spacing weaker, reversible, water-tight seal protein arrays on glass slides

9 MULTIPLEXED SCREENING PRTEIN MICRARRAYS functional genomics small molecule discovery protein profiling bottomless 384-well plate 64 wells per slide x 4 slides per plate = 256 wells per plate 256 wells per plate x 1 proteins per well = 25,6 assays per plate untreated cells ANTIBDY ARRAYS: SANDWIC ELISA treated cells 1. apply unlabeled protein 2. detect with labeled secondary antibody ANTIBDY ARRAYS: MICR-SANDWIC ASSAY Flourescent 2' Antibodies Antigen Capture Antibody lyse lyse fractionate fractionate anti- capture mab anti- capture mab anti- capture mab detected with mixture of: anti- mab#2-cy3 anti- mab#2-alexa488 anti- mab#2-cy5

10 A-431 squamous carcinoma MICR-SANDWIC DETECTIN TUMR CELL PRFILING micro-array flow cytometry ANTIBDY ARRAYS: MICR-SANDWIC ASSAY Ratiometric analyses of protein modification SK-BR-3 breast carcinoma P 4 P 4 P 4 fluorophore 25 5 antigen MCF-7 breast carcinoma 25 5 glass slide MCF-7 transfected PSPRYLATIN AND REGULATIN F GRWT FACTR RECEPTRS RATIMETRIC ANALYSES F EXPRESSIN AND PSPRYLATIN EGF Interrogate expression + Y-168 phosphorylation Interrogate expression + Y-1248 phosphorylation Interrogate expression (internal reference) P 4 P 4 α- α-py168 α- α-py1248 α- α- P 4 P 4 α- P 4 4 α- - is phosphorylated in response to EGF - is phosphorylated by - is down-regulated - remains unchanged Capture and detection antibodies against different epitopes Blue - Alexa488; Green - Cy3; Red - Cy5

11 P 4 P 4 P 4 P 4 RATIMETRIC PRFILING IN TUMR CELL LINES EFFECT F EGF EGF RATIMETRIC ANALYSES F EXPRESSIN AND PSPRYLATIN EGF time course on SK-BR-3 cells EGF anti- A-431 squamous carcinoma SK-BR-3 breast carcinoma MCF-7 breast carcinoma MCF-7 transfected anit- anti- EGF anti- anit- anti- RED = growth factor receptor expression GREEN = growth factor receptor phosphorylation BLUE = transferrin receptor expression YELLW = ratio expression/phosphorylation min 1 time of EGF treatment 6 min 12 min RATIMETRIC ANALYSES F EXPRESSIN AND PSPRYLATIN EGF time course on SK-BR-3 cells PRTEIN MICRARRAYS anti- receptor expression anti- Y168 receptor phosphorylation P4-Y168/ phoshorylation ratio functional genomics small molecule discovery protein profiling / py min 1 6 min 12 min min 1 6 min 12 min min 1 6 min 12 min anti- anti- Y1248 P4-Y168/ / py SUMMARY anti min 1 6 min 12 min min 1 6 min 12 min min 1 6 min 12 min rapid discovery of protein screening of small molecules in solution screening for specificity system-wide analysis at the protein level min 1 6 min 12 min

12 ACKNWLEDGEMENTS Protein Microarrays Prof. Peter Kim (Whitehead Institute) Dr. John Newman (Whitehead Institute) Dr. Viara Grantcharova Lioudmila Zaslavskaia Antibody Microarrays Prof. Peter Sorger (MIT) Dr. Ulrik Nielsen (MIT) Dr. Michael Cardone (MIT) Dr. Raghida Bu-Khalid arvard Center for Genomics Research DARPA