12/15/12
1
Dr. Sanjeeva Srivastava IIT Bombay
IIT Bombay Proteomics Course NPTEL
• Protein microarrays – an overview • Protein microarray experiment
• Applications • Biomarker screening
• Protein-protein interactions
2
12/15/12
2
IIT Bombay Proteomics Course NPTEL 3
DYNAMIC PROTEOME
Protein
Modification
Networks
Protein
Interaction
Networks
Genome Metabolome
Transcriptome Environmental
Factors
COMPREHENSIVE VIEW OF BIOLOGICAL PROCESSES
IIT Bombay Proteomics Course NPTEL 4
Reverse-phase MIST NAPPA Direct
Label-based Sandwich Chemical linkage Peptide fusion Abundance based Function based
12/15/12
3
IIT Bombay Proteomics Course NPTEL
IIT Bombay Proteomics Course NPTEL 6
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
12/15/12
4
IIT Bombay Proteomics Course NPTEL
IIT Bombay Proteomics Course NPTEL 8
Antibody Screens
Enzymatic Assays
Protein DNA
Protein Small
Molecule
Protein Lipid
Protein Protein
Cy3 Cy3
ATP ATP ADP
Cy3
Cy3
Cy3
Cy3
12/15/12
5
IIT Bombay Proteomics Course NPTEL
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
IIT Bombay Proteomics Course NPTEL
12/15/12
6
IIT Bombay Proteomics Course NPTEL
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
IIT Bombay Proteomics Course NPTEL 12
Sera from 30 cancer patients Sera from 30 healthy patients
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7
IIT Bombay Proteomics Course NPTEL 13
Microarray Data Analysis
Quality control
Autoantibody screening
Re-drawn from Hudson et al. 2007
IIT Bombay Proteomics Course NPTEL 14
Lamin A SSRP1 RALBP1 IRF6
MAGEB4
COIL NOB1P CBLB
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8
IIT Bombay Proteomics Course NPTEL 15
Serum Blocking reagent
Tissue
Chromogen
Lamin A Lamin C
p53
Healthy Tumor
Re-drawn from Hudson et al. 2007
IIT Bombay Proteomics Course NPTEL 16
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
Re-drawn from Hudson et al. 2007
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9
IIT Bombay Proteomics Course NPTEL 17
Detection
High Density Protein Microarrays
IIT Bombay Proteomics Course NPTEL
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
12/15/12
10
IIT Bombay Proteomics Course NPTEL 19
Inflammatory Bowel
Disease
E. coli Infection
Extraction & purification of E. coli proteins
E. coli protein chip
Biomarker detection
IIT Bombay Proteomics Course NPTEL 20
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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11
IIT Bombay Proteomics Course NPTEL 21
HC CD UC
169 18
185
6
38
1
HC
CD
UC
Re-drawn from Chen et al. 2009
IIT Bombay Proteomics Course NPTEL 22
**p-value < 0.0001 *p-value< 0.05
* * *
Re-drawn from Chen et al. 2009
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12
IIT Bombay Proteomics Course NPTEL 23
Healthy Controls
Crohn’s Disease
Ulcerative Colitis
Re-drawn from Chen et al. 2009
IIT Bombay Proteomics Course NPTEL
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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13
IIT Bombay Proteomics Course NPTEL 25
E. coli host cell N. meningitidis genes Cloned genes
Protein expression & purification
Array spotting Array screening
Detection & analysis
Purified bacterial proteins Patient sera
IIT Bombay Proteomics Course NPTEL
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
12/15/12
14
IIT Bombay Proteomics Course NPTEL 27
Polyacrlyamide-hydrogel slides
Prostate cancer patient sera
Control group sera
von Willebrand Factor
IgM Villin
IgG α 1-antichymotrypsin
IIT Bombay Proteomics Course NPTEL
12/15/12
15
IIT Bombay Proteomics Course NPTEL
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
IIT Bombay Proteomics Course NPTEL 30
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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16
IIT Bombay Proteomics Course NPTEL 31
Western blot
Re-drawn from Gong et al. 2008
IIT Bombay Proteomics Course NPTEL 32
Streptavidin
Biotin
Cy dye
Binding Protein or DNA
Immobilized TF Protein
Cy dye
Re-drawn from Gong et al. 2008
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17
IIT Bombay Proteomics Course NPTEL 33
At1g03800 At5g19790 At1g12980 At1g28160
At3g23220, At3g60490
EB
E
At4g16750
At4g25490
At1g63040
α-H
is
DB
E α-
His
E
BE
α-
His
D
BE
Re-drawn from Gong et al. 2008
IIT Bombay Proteomics Course NPTEL 34
12/15/12
18
IIT Bombay Proteomics Course NPTEL
Zhu, H. et al. Global analysis of protein activities using proteome chips. Science 2001, 293:2101-2105.
Case study-5
IIT Bombay Proteomics Course NPTEL 36
Nickel-coated slide
Array printing
His6 tagged yeast proteins
Biotinylated calmodulin
Phosphoinositides
Array signal
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19
IIT Bombay Proteomics Course NPTEL
IIT Bombay Proteomics Course NPTEL
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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20
IIT Bombay Proteomics Course NPTEL 39
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
IIT Bombay Proteomics Course NPTEL
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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21
IIT Bombay Proteomics Course NPTEL 41
Damaged DNA in nucleus
DNA damage Recognition
Expressed Mutated Proteins
Faulty Protein Activity
Protein Expression
E. coli
Re-drawn from Chen et al. 2008
IIT Bombay Proteomics Course NPTEL 42
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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22
IIT Bombay Proteomics Course NPTEL 43
kDa70
55
40
35
25
15
10
kDa70
55
40
35
25
15
10
Re-drawn from Chen et al. 2008
IIT Bombay Proteomics Course NPTEL 44
YbaZ
Anti-His Antibody Probe 1 Probe 2 Probe 3
Probe 4 Probe 5 Probe 6 Probe 7
Re-drawn from Chen et al. 2008
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23
IIT Bombay Proteomics Course NPTEL 45
Anti-His Antibody Probe 1 Probe 2 Probe 3
Probe 4 Probe 5 Probe 6 Probe 7
YbcN
Re-drawn from Chen et al. 2008
IIT Bombay Proteomics Course NPTEL
Ptacek et al. 2005, Global analysis of protein phosphorylation in yeast. Nature 438, 4187
Case study-8
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24
IIT Bombay Proteomics Course NPTEL 47
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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25
IIT Bombay Proteomics Course NPTEL 49
• An overview of protein microarray experiment • Applications
• Biomarker screening • Protein-protein interactions • Protein-DNA interaction • DNA damage study • Kinase substrate identification
IIT Bombay Proteomics Course NPTEL 50
• 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.
12/15/12
26
IIT Bombay Proteomics Course NPTEL 51
• 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.
IIT Bombay Proteomics Course NPTEL 52
• 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