This document discusses protein microarrays and their development and applications. It describes some key differences between protein and DNA microarrays, such as the challenges of amplifying and predicting protein activity and interactions due to their 3D structures. Various methods for capturing proteins on chips are presented, including different oriented immobilization techniques. Applications of protein microarrays include analyzing protein interactions, screening for drug targets, and developing techniques like self-assembly and covalent mRNA-protein fusion protein microarrays. Detection methods like fluorescence, enzymatic reactions, and mass spectrometry are also summarized.

1. Protein ChipProtein Chip
ProteinProtein MicroarrayMicroarray

2. Protein chipProtein chip vsvs DNA chipDNA chip
Not available yetNot available yetEstablished (PCR)Established (PCR)AmplificationAmplification
Not possible yet. Efforts are undertaken to predict modelsNot possible yet. Efforts are undertaken to predict models
that are based on sequence homologies, structure, etc.that are based on sequence homologies, structure, etc.
Well definedWell defined
Based on primary nucleotide sequenceBased on primary nucleotide sequence
Activity predictionActivity prediction
Very low to highVery low to high
Dependent on individual proteinDependent on individual protein
HighHighInteractionInteraction
specificityspecificity
Very low to highVery low to high
Dependent on individual protein:Dependent on individual protein:
HighHighInteraction affinityInteraction affinity
Multiple active interaction sitesMultiple active interaction sites1 by 1 interaction1 by 1 interactionInteraction sitesInteraction sites
33--D structure important for activity, avoid denaturationD structure important for activity, avoid denaturationDenatured, no loss of activity,Denatured, no loss of activity,
can be stored drycan be stored dry
Functional stateFunctional state
Individual typesIndividual types
Hydrophobic and/or hydrophilic domainsHydrophobic and/or hydrophilic domains
FragileFragile
UniformUniform
Hydrophilic acidic backboneHydrophilic acidic backbone
StableStable
StructureStructure
ProteinProteinDNADNAPropertiesProperties

3. Possible applications of proteinPossible applications of protein
microarraysmicroarrays

4. Two types of protein microarrays are categorized:
1. Analytical Microarray
2. Full-scale Genomic Protein Microarray

5. Schematic representation for strategy fabricatingSchematic representation for strategy fabricating
protein microarraysprotein microarrays

6. Importance of protein immobilizationImportance of protein immobilization

7. 101 ways of capture protein on chip101 ways of capture protein on chip
affibodies
antibodies
aptamers
Antibody sandwich
electrostatic
Van der Waals
Metal-chelate
Receptor-ligand
Protein-protein
Protein-DNA
Enzyme-substrate
Markus et al, Proteomics, 2003Markus et al, Proteomics, 2003

8. Molecules used in orientedMolecules used in oriented
immobilization of proteinsimmobilization of proteins
Proteins A, G, and LProteins A, G, and L
Biotin andBiotin and streptavidinstreptavidin
Molecules that recognize carbohydratesMolecules that recognize carbohydrates
NitrilotriaceticNitrilotriacetic acidacid
Nucleic acidNucleic acid

9. Single strandedSingle stranded oligonucleotidesoligonucleotides
((aptamersaptamers))AptusAptus –– fitfit
AptamersAptamers
Simple zinc ionsSimple zinc ions
Organic dye moleculesOrganic dye molecules
Substance PSubstance P
BacteriophageBacteriophage T4 DNA polymeraseT4 DNA polymerase
RibosomesRibosomes
RousRous sarcoma virusessarcoma viruses
Human red blood cell membranesHuman red blood cell membranes
Stability of theStability of the apatamerapatamer--ligandligand complex duringcomplex during
hybridization and washing procedureshybridization and washing procedures

10. Comparison of different protein capture agentsComparison of different protein capture agents

11. Toward optimized antibody microarrays: a comparisonToward optimized antibody microarrays: a comparison
of current microarray support material (I)of current microarray support material (I)
11 different array surfaces11 different array surfaces
Detection limit, interDetection limit, inter-- andand
intraintra--chip variation, storagechip variation, storage
characteristicscharacteristics
Result:Result:
PolyacrylamidePolyacrylamide--coated slidecoated slide
is more suitable for very lowis more suitable for very low
concentrations of antigenconcentrations of antigen
Individual exp. RequirementIndividual exp. Requirement
25 to 40,000 μmol per spot

13. Antigen arrayAntigen array:: Oriented immobilization of biologically activeOriented immobilization of biologically active
proteins as a tool for revealing protein interactions and functiproteins as a tool for revealing protein interactions and functionon

14. FullFull--scale genomic protein arrayscale genomic protein array:: GlobalGlobal
analysis of protein activities using proteome chips (I)analysis of protein activities using proteome chips (I)
Zhu et al, Science, 2001Zhu et al, Science, 2001
6566 Yeast genes.6566 Yeast genes.
Clone in expression vector: GSTClone in expression vector: GST--HisX6 (glutathione SHisX6 (glutathione S--
transferasetransferase polyhistidinepolyhistidine..
Expression and capture proteins by GST affinity.Expression and capture proteins by GST affinity.
65666566 protein samples representingprotein samples representing 58005800 (93.5% of total)(93.5% of total)
unique proteins were spotted in duplicate on a singleunique proteins were spotted in duplicate on a single
nickelnickel--coated microscope slide.coated microscope slide.
Detecting proteinDetecting protein--protein interaction:protein interaction: biotinylatedbiotinylated
calmodulincalmodulin in the presence of calcium.in the presence of calcium.

15. Global analysis of protein activities usingGlobal analysis of protein activities using
proteome chips (II)proteome chips (II) Zhu et al, Science, 2001Zhu et al, Science, 2001

16. Global analysis of protein activities usingGlobal analysis of protein activities using
proteome chips (III)proteome chips (III) Zhu et al, Science, 2001Zhu et al, Science, 2001

17. Self-assembly Protein Microarrays
Ramachandran et al.,2004, Science
B
target DNA
(plasmid)
Target
protein
monoclonal anti-
GST detection Ab
CA
biotin
Polyclonal
anti-GST
(capture Ab)
Add cell-free
expression system
AA.. Individually crossIndividually cross--linking plasmid DNA with biotin.linking plasmid DNA with biotin.
AA. Spotting plasmid DNA on chip.. Spotting plasmid DNA on chip.
BB. Expressing protein on chip using transcription/translation. Expressing protein on chip using transcription/translation coupling reaction.coupling reaction.
CC. Check rec. protein be attached on spot using. Check rec. protein be attached on spot using monclonedmoncloned AbAb..
CC. Examining the completion of rec. protein using. Examining the completion of rec. protein using AbAb against rec. proteinagainst rec. protein..

18. Self-assembly Protein Microarrays
Advantage:
direct expression of rec. protein from plasmid.
Disadvantage:
1. needs specific ab to check whether rec. protein has been
completely expressed.
2. because use of a common Ab to capture protein, it could
cause possible contamination after protein expression when
the reaction is carried out on chip-based.

19. CellCell--free protein expression (CFPE)free protein expression (CFPE)
Protein synthesis on aProtein synthesis on a
micromicro--chamber provideschamber provides
Short reaction time.Short reaction time.
Smaller amount ofSmaller amount of
expression vector.expression vector.
Allows easy handling ofAllows easy handling of
multiple samples inmultiple samples in
parallel.parallel.

20. On-spot Self-assembly Protein Microarrayer
A. Preparing chip surface (Z/X )
B. Using 3 in1 system of micro stamper separately delivery plasmid DNA on
spot
C. Performing transcription/translation reaction (reaction mixture will be
delivered from reservoir of 3 in1 system.
D. Check the completion and the presence of rec. protein using radioactive
kinase reaction.
Alan Lin
Genetic Institute
Natl. Yang-Ming Univ.
C
Z/X
target DNA
(plasmid)
Target
protein
RRVSA
RRVSA
X
phosphorylation
detection of rec.
protein
A D
surface
preparation
B
Add cell-free
expression system
XX X X X
H
is
ET-28ap
T7

21. On-spot Self-assembly Protein Microarrayer
All reactions are contained on spot because using 3 in 1 delivery
system.
Use an universal DNA binder (Z*) to capture plasmid.
On-spot plasmid amplification.
On-spot transcription.
On-spot translation.
On-spot self-attachment.
Use C-terminal RRVSA phosphorylation to confirm the
completion of protein expression.
The degree of phosphorylation is good for quantization.
Individual spot can be modified or treated in response to
functional studies– good for dynamic study and drug screening.
Z* , artificial DNA binding protein.

22. detection of rec.
protein by
[32
P]phosphorylation
quantity of
expression
Candidate
genes
x1
x2
x3
x4
x5
x6
x7
x8
treatment of inhibitor
(potential chemical drug)
chem.a
chem.b
chem.c
chem.d
chem.e
chem.f
chem.g
chem.h
detect protein
activity
Conc.
chem.a
chem.b
chem.c
chem.d
chem.e
chem.f
chem.g
chem.h
from chemical
library
Screening potential chemical drug using
On-spot Self-assembly Protein Microarray (OSSAP)

23. Nucleic acidNucleic acid
Polypeptides can be covalently linked toPolypeptides can be covalently linked to
their corresponding mRNA.their corresponding mRNA.
Enough sensitivity to detectEnough sensitivity to detect attomoleattomole
quantities of displayed protein withoutquantities of displayed protein without
signal amplification.signal amplification.
mRNA stability?mRNA stability?

24. Generating addressable protein microarrays withGenerating addressable protein microarrays with
PROfusionPROfusion covalent mRNAcovalent mRNA--protein fusionprotein fusion
technologytechnology WengWeng, et al, Proteomics 2002, et al, Proteomics 2002
Common 5Common 5’’ capture probe; common 3capture probe; common 3’’--capture probe; probecapture probe; probe
for FLAGfor FLAG epitopeepitope, HA11, HA11 epitopeepitope, MYC, MYC epitopeepitope..

25. aa
P
ATG
aa
aaaa
aa
P
ATG
aa
aa
aa
aa
aa
aaaa
aa
aaaa
aa
N
N
N
ATG5‘
5‘
5‘
mRNA-peptide complex
DNA
Prot./Prot.
interaction Antibody selection
Immobilized
Selection
Motif
dsDNA
5‘ 5‘
RTPCR
ProteinProtein microarraysmicroarrays with covalent mRNAwith covalent mRNA--protein fusion technologyprotein fusion technology
WengWeng, et al, Proteomics 2002, et al, Proteomics 2002

26. Generating addressable protein microarrays withGenerating addressable protein microarrays with
PROfusionPROfusion covalent mRNAcovalent mRNA--protein fusion technologyprotein fusion technology

27. aa
aa
aa
aaaa
aa
aa
aa
aaaa
aa
N
5‘
Antibody selection
dsDNA
5‘
anti-x-IgG
(Stop Translation)
Immobilization
RTPCR
Ribosome Display:
use for screening high-affinity therapeutic antibody

28. Abs andAbs and AbAb--like proteinslike proteins
Generated by a recombinant organism orGenerated by a recombinant organism or
byby in vitroin vitro translationtranslation
RecombinantRecombinant FabFab fragmentsfragments
SingleSingle--chain Abschain Abs
HelixHelix--stabilizedstabilized AbAb fragmentsfragments

30. TemplateTemplate--imprintedimprinted nanonano--structured surfacesstructured surfaces
for protein recognitionfor protein recognition Shi, et al, Nature, 1999Shi, et al, Nature, 1999

31. Analysis of proteomic chipAnalysis of proteomic chip
DetectionDetection
Choice of probe molecule(s)Choice of probe molecule(s)
Detection of the probe molecule(s)Detection of the probe molecule(s)

32. Choice of probe molecule(s)Choice of probe molecule(s)
Any molecule that might bind to or interact withAny molecule that might bind to or interact with
proteins can be used as probe for the new arrayproteins can be used as probe for the new array
They may be from different molecular classesThey may be from different molecular classes
(e.g., nucleic acid, polypeptides, or various type(e.g., nucleic acid, polypeptides, or various type
of ligands)of ligands)

33. DetectionDetection
FluorochromeFluorochrome--labeledlabeled AbAb/Laser scanning/Laser scanning
EnzymeEnzyme--linkedlinked AbAb/CCD camera/CCD camera
RadioisotopeRadioisotope--labeledlabeled analyte/phosphorimageranalyte/phosphorimager
Change of surface topology/atomic forceChange of surface topology/atomic force
microscopemicroscope
Other applicable techniquesOther applicable techniques

34. Summary of current detection methodsSummary of current detection methods
Electro-biochemic Mot necessary Electrophoretic movement Yes Medium/high

35. RadioisotopeRadioisotope--labeledlabeled analyte/phosphorimageranalyte/phosphorimager
MacBeathMacBeath, et al, Science, 2000, et al, Science, 2000
Spatial resolution:Spatial resolution:
1.2mm1.2mm
Printing Proteins asPrinting Proteins as
Microarrays for HighMicroarrays for High--
Throughput FunctionThroughput Function
DeterminationDetermination

36. Colorimetric detection of protein microarrays basedColorimetric detection of protein microarrays based
onon nanonano--gold probe coupled with silver enhancementgold probe coupled with silver enhancement
(I)(I) LiangLiang, et al, JIM, 2004, et al, JIM, 2004

37. Colorimetric detection of protein microarrays based onColorimetric detection of protein microarrays based on
nanogoldnanogold probe coupled with silver enhancement (II)probe coupled with silver enhancement (II)

38. Colorimetric detection of protein microarrays based onColorimetric detection of protein microarrays based on
nanogoldnanogold probe coupled with silver enhancement (III)probe coupled with silver enhancement (III)

39. PerspectivesPerspectives
Complete relational databases consisting ofComplete relational databases consisting of
information for temporal and spatial expressioninformation for temporal and spatial expression
profiles, as well as interaction profiles are essentialprofiles, as well as interaction profiles are essential
components of proteomicscomponents of proteomics
Standardized data structureStandardized data structure
SubstrateSubstrate
SurfaceSurface derivatizationderivatization
Proteins immobilizedProteins immobilized
Mode of immobilizationMode of immobilization
Printing devicePrinting device
Labeling reagentsLabeling reagents
AnalyteAnalyte characteristicscharacteristics
Detection deviceDetection device
Data analysis toolsData analysis tools

40. Screening Functional ProteomicsScreening Functional Proteomics
Platform Technology of 2Platform Technology of 2--DD
ProteomicProteomic MicrorrayMicrorray
Alan Lin
Institute of Genetics
National Yang-Mining University
Taipei, Taiwan

41. Chemical modification of 2Chemical modification of 2--D gelD gel
slabslab
IodoacetylIodoacetyl--LCLC--biotinbiotin
Biotin will be detectedBiotin will be detected
by fluorescenceby fluorescence--
conjugatedconjugated avidinavidin
Proteins can beProteins can be
oriented throughoriented through
biotin/biotin/avidinavidin
interaction to get ainteraction to get a
uniform orientationuniform orientation
Plate

42. RollingRolling--carpet slicingcarpet slicing
After modification, theAfter modification, the
entire 2Dentire 2D--gel will begel will be
sliced into thousandssliced into thousands
of gel cubesof gel cubes
Frozen with dryFrozen with dry--iceice
underneath during theunderneath during the
slicingslicing
Robot machine (Robot machine (EttanEttan
Spotter Picker)Spotter Picker)

43. Protein condensationProtein condensation
Design of an apparatus of columnDesign of an apparatus of column--typetype
electrode array for protein condensationelectrode array for protein condensation
Design of 2Design of 2--D extraction array for proteinD extraction array for protein
condensationcondensation

44. ColumnColumn--type electrode array fortype electrode array for
protein condensationprotein condensation
PolyampholytesPolyampholytes nature ofnature of
proteinprotein
Lower chamber has beenLower chamber has been
fabricated using MENSfabricated using MENS
techniquetechnique
Top view Side view
Top view Bottom view Side view
Upper chamber
Lower chamber

45. Design of 2Design of 2--D extraction array for proteinD extraction array for protein
condensationcondensation
DirectDirect electrophoreticelectrophoretic condensation of 2condensation of 2--DD
proteins to a microproteins to a micro--drop bydrop by electrowettingelectrowetting

46. 1. Micro Filling chip
2. Micro Array Stamper
3. Micro Bio Reaction Chip
Hand-Fill-in Reservoir
Micro-Fill-in Reservoir
Micro Stamper
Micro Needle connector
Bio-reaction surface
三合一晶片系統
曾繁根 教授
國立清華大學 工程與系統科學系

47. Protein Self-fill by
capillary force
Micro
Stamp
A B C D
微陣列壓印晶片
生醫反應晶片
三合一晶片系統
Primary Reservoir
生醫檢體充填晶片 Secondary
Reservoir
Membrane
√
√
國立清華大學 工程與系統科學系
曾繁根 教授

48. 第二代微填充晶片(9/30/2002)
6*6 wells
30 μm channel
曾繁根 教授
國立清華大學 工程與系統科學系

49. Detection of the probe molecule(s)Detection of the probe molecule(s)
Probes can be detectedProbes can be detected
based on their inherentbased on their inherent
characters (e.g.,characters (e.g.,
immunogenicity); beingimmunogenicity); being
labeled with a detectablelabeled with a detectable
tag (e.g., fluorescent,tag (e.g., fluorescent,
luminescent orluminescent or
radioactive monomersradioactive monomers
IodoacetylIodoacetyl--LCLC--biotin forbiotin for
modificationmodification -->>
aminopropyltrimethoxylsilaminopropyltrimethoxylsil
aneane and BSand BS33 treatedtreated
glass slideglass slide --> FITC> FITC--
conjugatedconjugated avidinavidin

50. Detection and analysis of the proteomicDetection and analysis of the proteomic
chipchip
Computer assisted (automated) detection andComputer assisted (automated) detection and
analysis of proteomic chipanalysis of proteomic chip
Identification of positiveIdentification of positive--reactive protein inreactive protein in
proteomic chip using MALDIproteomic chip using MALDI--toftof

51. Computer assisted (automated) detectionComputer assisted (automated) detection
and analysis of proteomic chipand analysis of proteomic chip
The data generated with fluorescent detectionThe data generated with fluorescent detection
can be read by a computerizedcan be read by a computerized ““readerreader”” oror
scanning for qualitative detection andscanning for qualitative detection and
quantitative measurement of the existences ofquantitative measurement of the existences of
protein speciesprotein species

52. Identification of positiveIdentification of positive--reactive protein inreactive protein in
proteomic chip using MALDIproteomic chip using MALDI--toftof
Protein responsible for positive assay fromProtein responsible for positive assay from
reference gelreference gel --> in gel digestion> in gel digestion --> MALDI> MALDI--toftof
Both peptide mass fingerprinting (PMF) andBoth peptide mass fingerprinting (PMF) and
internal sequencing will be carried outinternal sequencing will be carried out
Yield information about the identification of theYield information about the identification of the
proteinprotein

53. The application of 2DThe application of 2D--preteomicpreteomic
chipchip
Screening forScreening for disease biomarker proteinsdisease biomarker proteins
such as presuch as pre--cancerous marker proteins forcancerous marker proteins for
oral canceroral cancer
A discrimination analysis combined with aA discrimination analysis combined with a
blank test will validate the assortedblank test will validate the assorted
reactingreacting--antigen profileantigen profile
After a positive result is obtained, theAfter a positive result is obtained, the
individual proteins with a meaningfulindividual proteins with a meaningful
reaction will be identified by MALDIreaction will be identified by MALDI--toftof