Protein Chip Protein Microarray

Protein chip vs DNA chip

Properties
Structure

DNA
Uniform Hydrophilic acidic backbone Stable Denatured, no loss of activity, can be stored dry 1 by 1 interaction High High

Protein
Individual types Hydrophobic and/or hydrophilic domains Fragile 3-D structure important for activity, avoid denaturation Multiple active interaction sites Very low to high Dependent on individual protein: Very low to high Dependent on individual protein Not possible yet. Efforts are undertaken to predict models that are based on sequence homologies, structure, etc. Not available yet

Functional state Interaction sites Interaction affinity Interaction specificity Activity prediction Amplification

Well defined Based on primary nucleotide sequence Established (PCR)

Possible applications of protein microarrays

Two types of protein microarrays are categorized:

1. Analytical Microarray 2. Full-scale Genomic Protein Microarray

Schematic representation for strategy fabricating protein microarrays

Importance of protein immobilization

101 ways of capture protein on chip

Receptor-ligand

affibodies

electrostatic

Protein-protein antibodies

Protein-DNA Van der Waals

aptamers
Enzyme-substrate

Metal-chelate Antibody sandwich

Markus et al, Proteomics, 2003

Molecules used in oriented immobilization of proteins

Proteins A, G, and L Biotin and streptavidin Molecules that recognize carbohydrates Nitrilotriacetic acid Nucleic acid

Single stranded oligonucleotides (aptamers) Aptus fit

Aptamers
Simple zinc ions Organic dye molecules Substance P Bacteriophage T4 DNA polymerase Ribosomes Rous sarcoma viruses Human red blood cell membranes

Stability of the apatamer-ligand complex during hybridization and washing procedures

Comparison of different protein capture agents

Toward optimized antibody microarrays: a comparison of current microarray support material (I)
25 to 40,000 mol per spot

11 different array surfaces Detection limit, inter- and intra-chip variation, storage characteristics Result:
Polyacrylamide-coated slide is more suitable for very low concentrations of antigen Individual exp. Requirement

Antigen array: Oriented immobilization of biologically active

proteins as a tool for revealing protein interactions and function

Full-scale genomic protein array: Global

analysis of protein activities using proteome chips (I)
Zhu et al, Science, 2001

6566 Yeast genes. Clone in expression vector: GST-HisX6 (glutathione Stransferase polyhistidine. Expression and capture proteins by GST affinity. 6566 protein samples representing 5800 (93.5% of total) unique proteins were spotted in duplicate on a single nickel-coated microscope slide. Detecting protein-protein interaction: biotinylated calmodulin in the presence of calcium.

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

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

Self-assembly Protein Microarrays

Ramachandran et al.,2004, Science

A
target DNA (plasmid)
biotin

B
Polyclonal anti-GST (capture Ab)

Target protein

monoclonal antiGST detection Ab

Add cell-free expression system

A. A. B. C. C.

Individually cross-linking plasmid DNA with biotin. Spotting plasmid DNA on chip. Expressing protein on chip using transcription/translation coupling reaction. Check rec. protein be attached on spot using moncloned Ab. Examining the completion of rec. protein using Ab against rec. protein.

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.

Cell-free protein expression (CFPE)

Protein synthesis on a micro-chamber provides

Short reaction time. Smaller amount of expression vector. Allows easy handling of multiple samples in parallel.

On-spot Self-assembly Protein Microarrayer

A
surface preparation

B
target DNA (plasmid)

Target protein

phosphorylation detection of rec. protein

RRVSA

Z/X

RRVSA

X X

X X
Add cell-free expression system

XX

A. B. C. D.

Preparing chip surface (Z/X ) Using 3 in1 system of micro stamper separately delivery plasmid DNA on spot Performing transcription/translation reaction (reaction mixture will be delivered from reservoir of 3 in1 system. Check the completion and the presence of rec. protein using radioactive kinase reaction.
H is

T7
p ET-28a

Alan Lin Genetic Institute Natl. Yang-Ming Univ.

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.

Screening potential chemical drug using On-spot Self-assembly Protein Microarray (OSSAP)
Candidate genes x1 x2 x3 x4 x5 x6 x7 x8 detection of rec. protein by [32P]phosphorylation quantity of expression

treatment of inhibitor (potential chemical drug) Conc. chem.a chem.b chem.c chem.d chem.e chem.f chem.g chem.h detect protein activity chem.a chem.b chem.c chem.d chem.e chem.f chem.g chem.h

from chemical library

Nucleic acid
Polypeptides can be covalently linked to their corresponding mRNA. Enough sensitivity to detect attomole quantities of displayed protein without signal amplification. mRNA stability?

Generating addressable protein microarrays with PROfusion covalent mRNA-protein fusion technology Weng, et al, Proteomics 2002
Common 5 capture probe; common 3-capture probe; probe for FLAG epitope, HA11 epitope, MYC epitope.

Protein microarrays with covalent mRNA-protein fusion technology

Weng, et al, Proteomics 2002
dsDNA P DNA

ATG

aa aa aaaa N 5
ATG

N 5
ATG

aa aa aa aa aa aa aa aa aa aa aa aa N

mRNA-peptide complex

RTPCR
Immobilized Selection Motif Prot./Prot. interaction Antibody selection

Generating addressable protein microarrays with PROfusion covalent mRNA-protein fusion technology

Ribosome Display:
use for screening high-affinity therapeutic antibody
dsDNA

5 5
(Stop Translation)

aa aaaa aa aa aa aa aa aa N aa aa
Antibody selection

RTPCR
anti-x-IgG

Immobilization

Abs and Ab-like proteins

Generated by a recombinant organism or by in vitro translation
Recombinant Fab fragments Single-chain Abs Helix-stabilized Ab fragments

Template-imprinted nano-structured surfaces for protein recognition Shi, et al, Nature, 1999

Analysis of proteomic chip Detection

Choice of probe molecule(s) Detection of the probe molecule(s)

Choice of probe molecule(s)

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

Detection
Fluorochrome-labeled Ab/Laser scanning Enzyme-linked Ab/CCD camera Radioisotope-labeled analyte/phosphorimager Change of surface topology/atomic force microscope Other applicable techniques

Summary of current detection methods

Electro-biochemic

Mot necessary

Electrophoretic movement

Yes

Medium/high

Radioisotope-labeled analyte/phosphorimager
MacBeath, et al, Science, 2000

Spatial resolution: 1.2mm Printing Proteins as Microarrays for HighThroughput Function Determination

Colorimetric detection of protein microarrays based on nano-gold probe coupled with silver enhancement (I) Liang, et al, JIM, 2004

Colorimetric detection of protein microarrays based on nanogold probe coupled with silver enhancement (II)

Colorimetric detection of protein microarrays based on nanogold probe coupled with silver enhancement (III)

Perspectives
Complete relational databases consisting of information for temporal and spatial expression profiles, as well as interaction profiles are essential components of proteomics Standardized data structure
Substrate Surface derivatization Proteins immobilized Mode of immobilization Printing device Labeling reagents Analyte characteristics Detection device Data analysis tools

Screening Functional Proteomics

Platform Technology of 2-D Proteomic Microrray

Alan Lin Institute of Genetics National Yang-Mining University Taipei, Taiwan

Chemical modification of 2-D gel slab

Iodoacetyl-LC-biotin Biotin will be detected by fluorescenceconjugated avidin Proteins can be oriented through biotin/avidin interaction to get a uniform orientation

Plate

Rolling-carpet slicing
After modification, the entire 2D-gel will be sliced into thousands of gel cubes Frozen with dry-ice underneath during the slicing Robot machine (Ettan Spotter Picker)

Protein condensation
Design of an apparatus of column-type electrode array for protein condensation Design of 2-D extraction array for protein condensation

Column-type electrode array for protein condensation

Polyampholytes nature of protein Lower chamber has been fabricated using MENS technique
Top view Side view

Upper chamber

Lower chamber

Top view

Bottom view

Side view

Design of 2-D extraction array for protein condensation Direct electrophoretic condensation of 2-D proteins to a micro-drop by electrowetting

Hand-Fill-in Reservoir 1. Micro Filling chip

Micro-Fill-in Reservoir Micro Needle connector 2. Micro Array Stamper

Micro Stamper 3. Micro Bio Reaction Chip

Bio-reaction surface


Primary Reservoir

Secondary Reservoir

Membrane

Protein Self-fill by capillary force

Micro Stamp

(9/30/2002)

6*6 wells 30 m channel

Detection of the probe molecule(s)

Probes can be detected based on their inherent characters (e.g., immunogenicity); being labeled with a detectable tag (e.g., fluorescent, luminescent or radioactive monomers Iodoacetyl-LC-biotin for modification -> aminopropyltrimethoxylsil ane and BS3 treated glass slide -> FITCconjugated avidin

Detection and analysis of the proteomic chip

Computer assisted (automated) detection and analysis of proteomic chip Identification of positive-reactive protein in proteomic chip using MALDI-tof

Computer assisted (automated) detection and analysis of proteomic chip

The data generated with fluorescent detection can be read by a computerized reader or scanning for qualitative detection and quantitative measurement of the existences of protein species

Identification of positive-reactive protein in proteomic chip using MALDI-tof

Protein responsible for positive assay from reference gel -> in gel digestion -> MALDI-tof Both peptide mass fingerprinting (PMF) and internal sequencing will be carried out Yield information about the identification of the protein

The application of 2D-preteomic chip

Screening for disease biomarker proteins such as pre-cancerous marker proteins for oral cancer A discrimination analysis combined with a blank test will validate the assorted reacting-antigen profile After a positive result is obtained, the individual proteins with a meaningful reaction will be identified by MALDI-tof