12/15/12

Dr.SanjeevaSrivastava
IITBombay

Protein microarrays an overview

Protein microarray experiment

Applications
Biomarker screening
Protein-protein interactions

IIT Bombay

Proteomics Course

NPTEL

In Pro
Ne tera tein
tw ctio
or n
ks

M Pro
o
Ne dic tein
tw at
or ion
ks

12/15/12

DYNAMICPROTEOME

Metabolome

Genome

Environmental
Factors

Transcriptome

COMPREHENSIVEVIEWOFBIOLOGICALPROCESSES
IIT Bombay

Proteomics Course

Abundancebased

Direct
Label-based

IIT Bombay

Sandwich

NPTEL

Functionbased

Reverse-phase Chemical linkage

Proteomics Course

Peptide fusion

NAPPA

MIST

NPTEL

12/15/12

IIT Bombay

Proteomics Course

ORF

ORF
ORF

ORF

ORF

NPTEL

Expression Vector
(ORF Collections)

Assay performance

Protein Expression
(in vivo or in vitro)
ORF

Protein Purification

Slide

ORF

ORF

Expression Plasmid Construction and

Purification
IIT Bombay

ting

d
an
g
in
t
in o
Pr vitr ion
s
e
id in res
l
S
p
Ex

ORF
ORF

Prin

Proteomics Course

ProteinProtein
Interaction
Protein
Modication
Studies
ProteinDNA/
SmallMolecule
Interaction
NPTEL

12/15/12

IIT Bombay

Proteomics Course

NPTEL

Cy3
Antibody
Screens

Enzymatic
Assays

Protein
DNA

Protein
Small
Molecule

Cy3

ATP

ADP

ATP

Cy3

Cy3

Cy3

Protein
Lipid
Cy3

Protein
Protein
IIT Bombay

Proteomics Course

NPTEL

12/15/12

ProteinInterac8ons
Direct
Labelling

Cy3

ProteinModica8ons

An>body
Recogni>on

ProteinProtein
Interac8ons

ProteinSmall
MoleculeInterac8ons

ProteinNucleicacid
Interac8ons

Phosphoryla8on

Glycosyla8on

Ac

Acetyla8on

Ubiqui8na8on

IIT Bombay

Proteomics Course

NPTEL

IIT Bombay

Proteomics Course

NPTEL

12/15/12

Case study-1

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
IIT Bombay

Proteomics Course

Sera from 30 healthy patients

Sera from 30 cancer patients

IIT Bombay

12

NPTEL

Proteomics Course

NPTEL

12/15/12

Microarray
DataAnalysis

Quality control

Autoantibody
screening
Re-drawn from Hudson et al. 2007
IIT Bombay

13

Proteomics Course

NPTEL

LaminA

SSRP1

RALBP1

IRF6

MAGEB
4

COIL

NOB1P

CBLB

IIT Bombay

14

Proteomics Course

NPTEL

12/15/12

Healthy
Tumor

Lamin A
Lamin C
Chromogen

Blocking
reagent

Serum

p53

Tissue
Re-drawn from Hudson et al. 2007
IIT Bombay

15

Proteomics Course

NPTEL

Stage 2
Lamin A/C EOC

Stage 3
EOC

Stage 4
EOC

Positive Negative
Controls Controls

SSRP1

Stage 2
EOC

Stage 3
EOC

Stage 4
EOC

Positive Negative
Controls Controls

CA-125

Stage 2
EOC

Stage 3
EOC

Stage 4
EOC

Positive Negative
Controls Controls

Re-drawn from Hudson et al. 2007

IIT Bombay

16

Proteomics Course

NPTEL

12/15/12

Detection

High Density
Protein Microarrays
IIT Bombay

17

Proteomics Course

NPTEL

Case study-2

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

Proteomics Course

NPTEL

12/15/12

E. coli
Infection

Inflammatory
Bowel
Disease

Extraction & purification of

E. coli proteins

E. coli protein chip

Biomarker
detection
IIT Bombay

19

Proteomics Course

NPTEL

Immunogenic Spots
era
ybaN
yhgN
focA
gabT
ycdG
CD

HC

Crohns Disease (CD)

Healthy Control (HC)

Re-drawn from Chen et al. 2009

IIT Bombay

20

Proteomics Course

NPTEL

10

12/15/12

HC

CD

UC

HC
UC

169

18
38
1

185
CD
Re-drawn from Chen et al. 2009
IIT Bombay

21

Proteomics Course

NPTEL

*
*

**p-value < 0.0001

*p-value< 0.05
Re-drawn from Chen et al. 2009
IIT Bombay

22

Proteomics Course

NPTEL

11

12/15/12

Healthy
Controls

Crohns
Disease

Ulcerative
Colitis

Re-drawn from Chen et al. 2009

IIT Bombay

23

Proteomics Course

NPTEL

Case study-3

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

Proteomics Course

NPTEL

12

12/15/12

E. coli host cell

N. meningitidis
genes

Cloned genes

Array screening

Protein
expression &
purification

Array spotting

Purified bacterial
proteins

Patient sera

Detection &
analysis
IIT Bombay

25

Proteomics Course

NPTEL

Case study-4

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.
IIT Bombay

Proteomics Course

NPTEL

13

12/15/12

Prostate
cancer
patient sera

Control
group
sera

Polyacrlyamidehydrogel slides
IgM
von Willebrand
Factor

IIT Bombay

IIT Bombay

27

Villin

1-antichymotrypsin
IgG

Proteomics Course

NPTEL

Proteomics Course

NPTEL

14

12/15/12

Case study-5

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.

IIT Bombay

Proteomics Course

Transcription
Factor

NPTEL

Regulation
Gene

ACAGTG
A
Binding Site

Arabidopsis
thaliana

DNA-Protein
Interaction
Studies

IIT Bombay

30

Arabidopsis
thaliana gene

Construction of
Arabidopsis TF
ORFeome
Clones
L1 Gene

L2

Entry Clone

Protein-Protein
Interaction
Studies
Proteomics Course

Transformation to
Clones

NPTEL

15

12/15/12

Western blot

Re-drawn from Gong et al. 2008

IIT Bombay

31

Proteomics Course

NPTEL

Cy dye
Streptavidin

Cy dye

Biotin
Binding Protein
or DNA
Immobilized
TF Protein

Re-drawn from Gong et al. 2008

IIT Bombay

32

Proteomics Course

NPTEL

16

12/15/12

At5g19790

At1g12980

At1g28160

EBE

-His

At1g03800

At1g63040

At4g16750

DBE

-His

At3g23220, At3g60490

DBE

EBE

-His

At4g25490

Re-drawn from Gong et al. 2008

IIT Bombay

33

Proteomics Course

NPTEL

IIT Bombay

34

Proteomics Course

NPTEL

17

12/15/12

Case study-5

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

IIT Bombay

Proteomics Course

Array printing

NPTEL

Biotinylated
calmodulin

Phosphoinositides
Nickel-coated slide

IIT Bombay

36

His6 tagged yeast

proteins

Proteomics Course

Array signal

NPTEL

18

12/15/12

IIT Bombay

Proteomics Course

NPTEL

Case study-6

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).
IIT Bombay

Proteomics Course

NPTEL

19

12/15/12

Labelled
antibodies

Labelled antigens

`
Antibody array
1

Poly-L-lysine
coated slide

39

6
4
5
Varying antibody concentrations

6
4
5
Varying antigen concentrations
IIT Bombay

Antigen array

Proteomics Course

NPTEL

Case study-7

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

Proteomics Course

NPTEL

20

12/15/12

E. coli
Damaged DNA in nucleus

Expressed Mutated Proteins

Protein Expression

DNA damage
Recognition

Faulty Protein Activity

Re-drawn from Chen et al. 2008

IIT Bombay

41

Proteomics Course

NPTEL

Additon of lysis
buffer & Ni-NTA
Resin
Stock

Combine culture
Induce cells

Vortex

Transfer
Aliquot

Low-salt
Washing
Elute

High-salt
washing

Filter plates
Re-drawn from Chen et al. 2008

IIT Bombay

42

Proteomics Course

NPTEL

21

12/15/12

kDa
70

kDa
70

55

55

40

40

35

35

25

25

15

15

10

10

Re-drawn from Chen et al. 2008

IIT Bombay

43

Proteomics Course

Anti-His
Antibody

Probe 4

Probe 1

Probe 5

Probe 2

Probe 6

NPTEL

Probe 3

Probe 7

YbaZ
Re-drawn from Chen et al. 2008
IIT Bombay

44

Proteomics Course

NPTEL

22

12/15/12

Anti-His
Antibody

Probe 4

Probe 1

Probe 5

Probe 2

Probe 6

Probe 3

Probe 7

YbcN
Re-drawn from Chen et al. 2008
IIT Bombay

45

Proteomics Course

NPTEL

Case study-8

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

Proteomics Course

NPTEL

23

12/15/12

Overexpress
and purify
kinases

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

Protein Chip
ATP

30C

ADP

Wash

Expose to film

Kinase substrate Identification

IIT Bombay

47

Proteomics Course

IIT Bombay

48

Proteomics Course

NPTEL

Modified from Ptacek et al. 2005

NPTEL

24

12/15/12

An overview of protein microarray experiment

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

IIT Bombay

49

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
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.

IIT Bombay

50

Proteomics Course

NPTEL

25

12/15/12

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 717730,
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 1419.

IIT Bombay

51

Proteomics Course

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

IIT Bombay

52

Proteomics Course

NPTEL

NPTEL

26