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 mol per
spot
12. 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
13. 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.
14. 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
15. 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
16. 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..
17. 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.
18. 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.
19. 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
20. 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.
21. 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)
22. 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?
23. 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..
24. 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
25. Generating addressable protein microarrays withGenerating
addressable protein microarrays with PROfusionPROfusion covalent
mRNAcovalent mRNA--protein fusion technologyprotein fusion
technology
26. 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
27. 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
28. TemplateTemplate--imprintedimprinted nanonano--structured
surfacesstructured surfaces for protein recognitionfor protein
recognition Shi, et al, Nature, 1999Shi, et al, Nature, 1999
29. 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)
30. 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)
31. 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
32. Summary of current detection methodsSummary of current
detection methods Electro-biochemic Mot necessary Electrophoretic
movement Yes Medium/high
33. 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
34. 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
35. 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)
36. 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)
37. 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
38. 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
39. 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
40. 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)
41. 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
42. 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
43. 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
45. Protein Self-fill by capillary force Micro Stamp A B C D
Primary Reservoir Secondary Reservoir Membrane
46. (9/30/2002) 6*6 wells 30 m channel
47. 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
48. 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
49. 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
50. 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
51. 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
