Protein microarrays – an overview Applications

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Dr. Sanjeeva Srivastava IIT Bombay

IIT Bombay Proteomics Course NPTEL

•  Protein microarrays – an overview •  Protein microarray experiment

•  Applications •  Biomarker screening

•  Protein-protein interactions

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IIT Bombay Proteomics Course NPTEL 3

DYNAMIC PROTEOME

Protein

Modification

Networks

Protein

Interaction

Networks

Genome Metabolome

Transcriptome Environmental

Factors

COMPREHENSIVE VIEW OF BIOLOGICAL PROCESSES


Reverse-phase MIST NAPPA Direct

Label-based Sandwich Chemical linkage Peptide fusion Abundance based Function based

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Expression Vector (ORF Collections)

Protein Expression (in vivo or in vitro)

ORF

ORF

ORF

ORF

ORF

Protein Purification Slide Printing

Assay performance

Expression Plasmid Construction and Purification

Slide Printin

g and

in vit

ro

Expressi

on

ORF

ORF ORF

ORF

ORF

Protein –Protein Interaction

Protein Modification

Studies

Protein –DNA / Small Molecule Interaction

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Antibody Screens

Enzymatic Assays

Protein DNA

Protein Small

Molecule

Protein Lipid

Protein Protein

Cy3 Cy3

ATP ATP ADP

Cy3

Cy3

Cy3

Cy3

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Protein Modifica8ons Protein Interac8ons

Phosphoryla8on Glycosyla8on

Ubiqui8na8on Acetyla8on

Protein‐Small Molecule Interac8ons

Protein‐Protein Interac8ons

Protein‐Nucleic acid Interac8ons

Cy3

Direct Labelling An>body

Recogni>on P

Ac U


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Hudson et al. Identification of differentially expressed proteins in ovarian cancer using high-density protein microarrays. Proc Natl Acad Sci USA. 2007, 104, 17494-9

Case study-1


Sera from 30 cancer patients Sera from 30 healthy patients

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Microarray Data Analysis

Quality control

Autoantibody screening

Re-drawn from Hudson et al. 2007


Lamin A SSRP1 RALBP1 IRF6

MAGEB4

COIL NOB1P CBLB

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Serum Blocking reagent

Tissue

Chromogen

Lamin A Lamin C

p53

Healthy Tumor



Stage 2 EOC

Stage 3 EOC

Stage 4 EOC

Negative Controls

Positive Controls Lamin A/C

Stage 2 EOC

Stage 3 EOC

Stage 4 EOC

Negative Controls

Positive Controls SSRP1

Stage 2 EOC

Stage 3 EOC

Stage 4 EOC

Negative Controls

Positive Controls CA-125


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Detection

High Density Protein Microarrays


Chen et al. Identification of novel serological biomarkers for inflammatory bowel disease using Escherichia coli proteome chip Mol Cell Proteomics. 2009, 8, 1765

Case study-2

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Inflammatory Bowel

Disease

E. coli Infection

Extraction & purification of E. coli proteins

E. coli protein chip

Biomarker detection


CD HC

era ybaN

yhgN focA

gabT ycdG

Immunogenic Spots

Crohn’s Disease (CD) Healthy Control (HC) Re-drawn from Chen et al. 2009

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HC CD UC

169 18

185

6

38

1

HC

CD

UC

Re-drawn from Chen et al. 2009


**p-value < 0.0001 *p-value< 0.05

* * *


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Healthy Controls

Crohn’s Disease

Ulcerative Colitis



Steller, S. et al. Bacterial protein microarrays for identification of new potential diagnostic markers for Neisseria meningitidis infections. Proteomics 2005, 5, 2048-2055.

Case study-3

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E. coli host cell N. meningitidis genes Cloned genes

Protein expression & purification

Array spotting Array screening

Detection & analysis

Purified bacterial proteins Patient sera


Miller JC, Zhou H, Kwekel J, Cavallo R, Burke J, Butler EB, Teh BS, Haab BB: Antibody microarray profiling of human prostate cancer sera: antibody screening and identification of potential biomarkers. Proteomics 2003, 3:56-63.

Case study-4

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Polyacrlyamide-hydrogel slides

Prostate cancer patient sera

Control group sera

von Willebrand Factor

IgM Villin

IgG α 1-antichymotrypsin


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Gong W et al The development of protein microarrays and their applications in DNA-protein and protein-protein interaction analyses of Arabidopsis transcription factors. Mol Plant. 2008, 1, :27-41.

Case study-5


Protein-Protein Interaction

Studies

DNA-Protein Interaction

Studies

Arabidopsis thaliana

Transcription Factor

ACAGTGA

Gene

Binding Site

Regulation

Arabidopsis thaliana gene Construction of

Arabidopsis TF ORFeome

Clones

Entry Clone Transformation to

Clones

L1 Gene L2

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Western blot

Re-drawn from Gong et al. 2008


Streptavidin

Biotin

Cy dye

Binding Protein or DNA

Immobilized TF Protein

Cy dye


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At1g03800 At5g19790 At1g12980 At1g28160

At3g23220, At3g60490

EB

E

At4g16750

At4g25490

At1g63040

α-H

is

DB

E α-

His

E

BE

α-

His

D

BE



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Zhu, H. et al. Global analysis of protein activities using proteome chips. Science 2001, 293:2101-2105.

Case study-5


Nickel-coated slide

Array printing

His6 tagged yeast proteins

Biotinylated calmodulin

Phosphoinositides

Array signal

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Haab B, Dunham M, Brown P: Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions. Genome Biol 2001, 2 (2).

Case study-6

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Poly-L-lysine coated slide

Antibody array

Labelled antigens

Antigen array

Labelled antibodies

`

1 2 3

4 5 6 Varying antigen concentrations

1 2 3

4 5 6 Varying antibody concentrations


Chen et al. A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli. Nat Methods 2008, 5, 69-74

Case study-7

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Damaged DNA in nucleus

DNA damage Recognition

Expressed Mutated Proteins

Faulty Protein Activity

Protein Expression

E. coli



Stock Combine culture

Induce cells

Additon of lysis buffer & Ni-NTA

Resin

Vortex

Filter plates

High-salt washing

Low-salt Washing

Aliquot

Transfer

Elute Re-drawn from Chen et al. 2008

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kDa70

55

40

35

25

15

10

kDa70

55

40

35

25

15

10



YbaZ

Anti-His Antibody Probe 1 Probe 2 Probe 3

Probe 4 Probe 5 Probe 6 Probe 7


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Anti-His Antibody Probe 1 Probe 2 Probe 3

Probe 4 Probe 5 Probe 6 Probe 7

YbcN



Ptacek et al. 2005, Global analysis of protein phosphorylation in yeast. Nature 438, 4187

Case study-8

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Protein Chip

Kinase substrate Identification

ATP ADP 30C

Incubate with kinase and [Y-33p]-ATP

Overexpress and purify

kinases

Wash

Expose to film

IIT Bombay Proteomics Course NPTEL 48 Modified from Ptacek et al. 2005

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•  An overview of protein microarray experiment •  Applications

•  Biomarker screening •  Protein-protein interactions •  Protein-DNA interaction •  DNA damage study •  Kinase substrate identification


•  Hudson et al. Identification of differentially expressed proteins in ovarian cancer using high-density protein microarrays. Proc Natl Acad Sci USA. 2007, 104, 17494-9

•  Chen et al. Identification of novel serological biomarkers for inflammatory bowel disease using Escherichia coli proteome chip Mol Cell Proteomics. 2009, 8, 1765

•  Steller, S. et al. Bacterial protein microarrays for identification of new potential diagnostic markers for Neisseria meningitidis infections. Proteomics 2005, 5, 2048-2055.

•  Miller JC, Zhou H, Kwekel J, Cavallo R, Burke J, Butler EB, Teh BS, Haab BB: Antibody microarray profiling of human prostate cancer sera: antibody screening and identification of potential biomarkers. Proteomics 2003, 3:56-63.

•  Gong W et al The development of protein microarrays and their applications in DNA-protein and protein-protein interaction analyses of Arabidopsis transcription factors. Mol Plant. 2008, 1, :27-41.

•  Zhu, H. et al. Global analysis of protein activities using proteome chips. Science 2001, 293:2101-2105.

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•  Haab B, Dunham M, Brown P: Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions. Genome Biol 2001, 2 (2).

•  Chen et al. A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli. Nat Methods 2008, 5, 69-74

•  Ptacek et al. 2005, Global analysis of protein phosphorylation in yeast. Nature 438, 4187

•  Harini Chandra, Sanjeeva Srivastava. 2010. Cell-free synthesis-based protein microarrays and their applications. PROTEOMICS. Volume 10, Issue 4, pages 717–730, No. 4 February 2010

•  Harini Chandra, Panga Jaipal Reddy and Sanjeeva Srivastava. 2011. Expert Review of Proteomics. Protein microarrays and novel detection platforms. February 2011, Vol. 8, No. 1, Pages 61-79 10.1586/epr.10.99

•  Joshua LaBaer , Niroshan Ramachandran. 2005. Protein microarrays as tools for functional proteomics. Current Opinion in Chemical Biology. Volume 9, Issue 1, February 2005, Pages 14–19.


•  Wei Huang, Lin Wu, Guozhen Liu, Siqi Liu. Protein microarray: A key approach of proteomics, .August 2010, Volume 5, Issue 4, pp 331-338

•  Wei Huang, Lin Wu, Guozhen Liu, Siqi Liu. Protein microarray: A key approach of proteomics. Frontiers in Biology, August 2010, Volume 5, Issue 4, pp 331-338

Documents

Protein microarrays – an overview Applications