Extending Microarray Technology print head to Study Protein - - PowerPoint PPT Presentation

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Extending Microarray Technology print head to Study Protein - - PowerPoint PPT Presentation

Oct 01, 2022 328 likes •464 views

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 m Gavin MacBeath split pins Bauer Center for Genomics Research

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SLIDE 1

Extending Microarray Technology to Study Protein Function

Gavin MacBeath Bauer Center for Genomics Research Harvard University MICROARRAYS: THE POWER OF MULTIPLEXING

Samples in 96-

  • r 384-well plates

Slit width: ~ 30 µm

arrayer 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-

  • r 384-well plates

Slit width: ~ 30 µm

arrayer 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
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SLIDE 2

OH OH OH EtO Si OEt OEt H2N

(1)

Glass Slide

(2)

N O O N O O O O O

(4)

N O O N O O O O O

BSA-NHS Slide

(3)

BSA

O O O Si Si Si O O OH OH H N NH2 H N O O H N H N O O N O O H N O O N O O

BSA

O O O Si Si Si O O OH OH H N NH2 H N O O H N H N O H N O

BSA

H N H N CO2

  • H2N

H2N CO2

  • (2)

(1)

A B BSA-NHS SLIDES

OH OH OH

Glass Slide Aldehyde Slide

EtO Si OEt OEt O H H2N O Si O Si O OH O H O H O Si O OH O H N O Si O Si O OH N H H O Si O OH H

BSA

N H2N

BSA

H2N

(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

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SLIDE 3

p50 GST-FRB 0.50 µg/ml BODIPY-FL-IgG 0.05 µg/ml Cy3-IΚBα 0.50 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-IgG 0.50 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) Fluorescence units GST-FRB

2000 µg/ml 1000 µg/ml 500 µg/ml 150 pg/ml (12.5 pM)

PROTEIN MICROARRAYS protein- protein interactions enzyme- substrate interactions protein- small molecule interactions enzyme- substrate interactions

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SLIDE 4

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

Fluorophore

BSA

Small Molecule

BSA

Glass Slide

PROTEIN-SMALL MOLECULE INTERACTIONS

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SLIDE 5

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 functional genomics protein profiling small molecule discovery subsets of related proteins protein domains full-length proteins

  • informatics (sequence motifs)
  • literature searches
  • coiled coils

John Newman

  • protocadherins

Viara Grantcharova

  • cancer proteins

Joshua LaBaer, Pascal Braun

  • c. elegans development

Viara 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 tropomyosin homodimer myc/max heterodimer

J Mol Biol (1998) 281, 165. J Mol Biol (1986) 192, 111.

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SLIDE 6

COILED-COIL SCREEN: YEAST TWO-HYBRID Pilot Screen

Newman, Wolf & Kim (2000) PNAS 97, 13203 DNA Binding Domain Constructs Activation Domain Constructs

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

  • Met28p

Gcn4p Met4p Spc42p Max Mad Myc Mxi1 John Newman

Proteins 1-72

Cy3-Met28p / Met4p Cy3-Spc42p / Spc42p

PILOT COILED-COIL SCREEN: PROTEIN MICROARRAYS

John Newman

functional genomics protein profiling PROTEIN MICROARRAYS small molecule discovery

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SLIDE 7

SCREENING METHOD 2 protein microarrays SCREENING METHOD 2

wash and scan

protein microarrays SCREENING METHOD 2 protein microarrays SCREENING METHOD 2 protein microarrays small molecule library

each well contains all the labeled ligands and a different small molecule

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SLIDE 8

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 bottomless 384-well plate protein arrays

  • n glass slides

strong adhesive weaker, reversible, water-tight seal

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SLIDE 9

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

protein profiling functional genomics PROTEIN MICROARRAYS small molecule discovery ANTIBODY ARRAYS: SANDWICH ELISA

untreated cells lyse lyse fractionate fractionate treated cells

  • 1. apply unlabeled protein
  • 2. detect with labeled

secondary 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

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SLIDE 10

TfR ErbB2 EGFR 1000 3000 2000 TfR ErbB2 EGFR 1000 3000 2000 TfR ErbB2 EGFR 1000 3000 2000 TfR ErbB2 EGFR 1000 3000 2000 250 500 250 500 250 500 250 500

A-431 squamous carcinoma SK-BR-3 breast carcinoma MCF-7 transfected ErbB2 MCF-7 breast carcinoma micro-array flow cytometry

MICRO-SANDWICH DETECTION TUMOR CELL PROFILING

glass slide

ANTIBODY ARRAYS: MICRO-SANDWICH ASSAY

antigen

PO

4

PO

4

PO

4

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

EGFR EGFR

EGFR EGFR ErbB2 TfR EGF

PHOSPHORYLATION AND REGULATION OF GROWTH FACTOR RECEPTORS

P O 4 PO 4

RATIOMETRIC ANALYSES OF EXPRESSION AND PHOSPHORYLATION

Blue - Alexa488; Green - Cy3; Red - Cy5 Capture and detection antibodies against different epitopes Interrogate TfR expression (internal reference)

TfR α-TfR α-TfR

Interrogate ErbB2 expression + Y-1248 phosphorylation

ErbB2

4

α-ErbB2

PO 4

α-ErbB2 α-pY1248

Interrogate EGFR expression + Y-1068 phosphorylation

EGFR

PO

4

α-EGFR

PO 4

α-EGFR α-pY1068

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SLIDE 11

EFFECT OF EGF

RATIOMETRIC PROFILING IN TUMOR CELL LINES

RED = growth factor receptor expression GREEN = growth factor receptor phosphorylation BLUE = transferrin receptor expression YELLOW = ratio expression/phosphorylation

anti-EGFR anit-ErbB2 anti-TfR EGF A-431 squamous carcinoma

+

  • SK-BR-3

breast carcinoma

+

  • MCF-7

breast carcinoma

+

  • MCF-7

transfected ErbB2

+

  • EGFR

EGFR ErbB2 TfR EGF

P O 4 PO 4

RATIOMETRIC ANALYSES OF EXPRESSION AND PHOSPHORYLATION

EGF time course on SK-BR-3 cells TfR

EGFR EGFR

EGFR EGFR ErbB2 EGF

P O 4 PO 4

time of EGF treatment anti-EGFR anit-ErbB2 anti-TfR 0 min 1 min 5 min 15 min 60 min 120 min

0 min 1 min 5 min 15 min 60 min 120 min 100 200 300 400 500 anti-EGFR Y1068 300 600 900 1200 anti-EGFR 0 min 1 min 5 min 15 min 60 min 120 min 0.1 0.2 0.3 0.4 0.5 0 min 1 min 5 min 15 min 60 min 120 min PO4-Y1068/ EGFR anti-ErbB2 Y1248 250 500 750 1000 0 min 1 min 5 min 15 min 60 min 120 min 1000 2000 3000 4000 anti-ErbB2 0 min 1 min 5 min 15 min 60 min 120 min PO4-Y1068/ ErbB2 0.1 0.2 0.3 0.4 0 min 1 min 5 min 15 min 60 min 120 min

RATIOMETRIC ANALYSES OF EXPRESSION AND PHOSPHORYLATION

EGF time course on SK-BR-3 cells

EGFR/ pY1068 ErbB2/ pY1248

phoshorylation ratio

anti-TfR 1000 2000 3000 0 min 1 min 5 min 15 min 60 min 120 min

TfR

receptor expression receptor phosphorylation

functional genomics protein profiling

  • 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 molecule discovery

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SLIDE 12

ACKNOWLEDGEMENTS

Antibody Microarrays

  • Prof. Peter Sorger (MIT)
  • Dr. Ulrik Nielsen (MIT)
  • Dr. Michael Cardone (MIT)
  • Dr. Raghida Bu-Khalid

Harvard Center for Genomics Research DARPA Protein Microarrays

  • Prof. Peter Kim (Whitehead Institute)
  • Dr. John Newman (Whitehead Institute)
  • Dr. Viara Grantcharova

Lioudmila Zaslavskaia