Biotech 2012 spring-2-protein_chips

Protein Arrays (Biosurfaces for Proteome

Research)

Outline

Protein Analysis – Introduction Why ? How ?

New Protein Analysis Tools Protein Arrays SELDI MS – Based ProteinChip®

DNA

mRNA

Protein

Central Dogma of Life

Protection of DNA

Amplification of genetic information

Efficient regulation of gene expression

Proteins

20 amino acids

30,000 – 34,000 genes

2,000,000 proteins

Protein Functions

Signal transduction

Transcription regulation

Immune response

Other vital cellular actions

Proteomics An organism’s proteome:

a catalog of all proteins expressed throughout life expressed under all conditions

The goals of proteomics: to catalog all proteins to understand their functions to understand how they interact with

each other

Gel electrophoresis, northern/western blot (fluorescence/radio active label)

X-ray crystallography

2D - mass spectrometry

Protein microarrays

Antibody Array for Protein Antibody Array for Protein Expression Profiling Expression Profiling

Methods for Protein AnalysisMethods for Protein Analysis

1. High throughput analysis of hundreds of thousands of proteins.

2. Proteins are immobilized on glass chip.

3. Various probes (protein, lipids, DNA, peptides, etc) are used.

Part1

Protein Microarray

Protein Array VS DNA Microarray

Target: Proteins DNA(Big, 3D) (Small, 2D)

Binding: 3D affinity 2D seqStability: Low HighSurface: Glass GlassPrinting: Arrayer ArrayerAmplification: Cloning PCR

Protein Array Fabrication

Protein substratesProtein substrates Polyacrylamide or Polyacrylamide or

agarose gelsagarose gels GlassGlass NanowellsNanowells

Proteins deposited Proteins deposited on chip surface by on chip surface by robotsrobots

Benfey & Protopapas, 2005

Protein Attachment


Diffusion Protein suspended in

random orientation, but presumably active

Adsorption/Absorption Some proteins inactive

Covalent attachment Some proteins inactive

Affinity Orientation of protein

precisely controlled

Diffusion

Adsorption/Absorption

Covalent

Affinity

Protein Interactions


Different capture molecules must be used to study different interactions

Examples Antibodies (or antigens) for

detection Proteins for protein-protein

interaction Enzyme-substrate for

biochemical functionReceptor–

ligand

Antigen–antibody

Protein–protein

Aptamers

Enzyme–substrate

Expression Array Probes (antibody) on surface recognize

target proteins.

Identification of expressed proteins from samples.

Typical quantification method for large # of expressed proteins.

Interaction Array Probes (proteins, peptides, lipids) on

surface interact with target proteins.

Identification of protein interactions.

High throughput discovery of interactions.

Functional Array Probes (proteins) on surface react with

target molecules .

Reaction products are detected.

Main goal of proteomics.

DetectionDetection The preferred method of detection currently is The preferred method of detection currently is

fluorescence detection. The fluorescent detection. The fluorescent detection method is compatible with standard detection method is compatible with standard microarray scanners, the spots on the resulting microarray scanners, the spots on the resulting image can be quantified by commonly used image can be quantified by commonly used microarray quantification software packages. microarray quantification software packages. However, some minor alterations to the analysis However, some minor alterations to the analysis software may be needed. Other common software may be needed. Other common detection methods include colorimetric detection methods include colorimetric techniques based on silver-precipitation, techniques based on silver-precipitation, chemiluminescent and label free Surface chemiluminescent and label free Surface Plasmon Resonance.Plasmon Resonance.

ResourcesResources

http://www.microarraystation.com/http://www.microarraystation.com/

Technical Challenges in Protein Chips

1. Poor control of immobilized protein activity.

2. Low yield immobilization.

3. High non-specific adsorption.

4. Fast denaturation of Protein.

5. Limited number of labels – low mutiplexing

“Global Analysis of Protein Activities Using Proteome Chips”

Snyder Lab, Yale University2101-2105, Vol 293, Science, 2001

Objectives

1.Construct yeast proteome chip containing 80% of yeast proteins in high throughput manner.

2.Study protein interactions at cell level using the proteome chip.


Snyder Lab, Yale University, 2101-2105, Vol 293, Science, 2001

Protein Immobilization on Surface

1. Cloning of 5800 ORFs.

2. Production of fusion proteins (GST- HisX6).

3. Printing on glass chip.

4. Verification by anti-GST.



Protein-Protein Interactions

1. Calmodulin-Biotin with Ca++.

2. Interaction checked with Cy-3-streptavidin

3. Six calmodulin targets newly found.

4. Another six known targets could not be detected.



Protein-Lipid Interactions

1. Phospholipids-Biotin.

2. About 150 proteins interacted with phospholipid probes.

3. Several of them were un-known, and some related to glucose metabolism.



Conclusions

1. Novel tool for protein interaction studies.

2. Concerns : * indirect interaction?

* missing proteins?

* surface chemistry?



Antibody Array for Antibody Array for Protein Expression Protein Expression

Profiling Profiling http://www.youtube.com/watch?v=E

eiN6bebCEw

SELDI MS-based ProteinChip

Utilizes Surface Enhanced Laser Desorption/Ionization Mass Spectrometry (1993)

MALDI MS combined with chromatography (Bioaffinity): surface-MALDI

Part2

3) Energy absorbing 3) Energy absorbing molecules are added to molecules are added to retained proteins. retained proteins. Following laser desorption Following laser desorption and ionization of proteins, and ionization of proteins, Time-of Flight (TOF) mass Time-of Flight (TOF) mass spectrometry accurately spectrometry accurately determines their massesdetermines their masses

Protein Analysis by SELDI-MS

Source:http://dir.niehs.nih.gov/proteomics/emerg3.htm

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2

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1) Apply sample (serum, 1) Apply sample (serum, tissue extract, etc.) to tissue extract, etc.) to ProteinChip® array.ProteinChip® array.

2) Wash sample with increasing 2) Wash sample with increasing stringency to remove non-specific stringency to remove non-specific proteins.proteins.

Advantages & Applications of SELDI MS Extraction, fractionation, clean-up and amplification of

samples on surface High throughput, high level multiplexing Large scale/ Low sample volume High sensitivity Various molecules on surface to capture probes Discover protein biomarkers Purification of target proteins Other fundamental proteomics research

Mass Spectrometry

Mass Spectrometry : Components

1. Ion source – sample molecules are ionized.

Chemical, Electrospray, Matrix-assisted laser desorption ionization

2. Mass analyzer – ions are separated based on their masses.

Time-of-flight, Quadruple, Ion trap

3. Mass detector

4. Data acquisition units

Ion Sources Proteomics requires

specialized ion sources Electrospray Ionization

(ESI) With capillary

electrophoresis and liquid chromatography

Matrix-assisted laser desorption/ionization (MALDI) Extracts ions from sample

surface

ESI

MALDI


Mass Analyzer


Ion trap Captures ions on the

basis of mass-to-charge ratio

Often used with ESI Time of flight (TOF)

Time for accelerated ion to reach detector indicates mass-to-charge ratio

Frequently used with MALDI

Also other possibilities

Ion Trap

Time of Flight

Detector

Mass Spectrometry for Proteins

1. ESI-Ion Trap

Sample in solution, lower mass limit.

2. MALDI-TOF

Solid state measurement, high mass limit, most popular tool for protein analysis.

Protein Identification by MS Preparation of protein samplePreparation of protein sample

Extraction from a gelExtraction from a gel Digestion by proteases — e.g., trypsinDigestion by proteases — e.g., trypsin

Mass spectrometer measures mass-charge ratio of Mass spectrometer measures mass-charge ratio of peptide fragmentspeptide fragments

Identified peptides are compared with databaseIdentified peptides are compared with database Software used to generate theoretical peptide Software used to generate theoretical peptide

mass fingerprint (PMF) for all proteins in databasemass fingerprint (PMF) for all proteins in database Match of experimental readout to database PMF Match of experimental readout to database PMF

allows researchers to identify the proteinallows researchers to identify the protein

Mass Spectrum of Protein mixture

Advantages of Mass Spectrometry

1. No labeling required.

2. Fast separation.

3. Multiplexing feasibility.

4. High sensitivity.

Disadvantages of Mass Spectrometry

1. Lower sensitivity compared to array.

2. Lower accuracy in quantitative assay.

3. Stringent sample purity.

“SELDIProteinChip Array Technology: Protein-Based Predictive Medicine and

Drug Discovery Applications”

Ciphergen Biosystems, Inc, 237-241, Vol 4, J. Biomed. & Biotechnol., 2003

“SELDIProteinChip Array Technology: Protein-Based Predictive Medicine and Drug Discovery Applications”


SELDIProteinChip Array Technology

1. ProteinChip Array, ProteinChip Reader, asso. software

2. Surface: hydrophobic, hydrophilic, ion exchange, metal-immobilized, etc…

3. Probes (baits): antibodies, receptors, oligonucleotides

4. Samples: cell lysates, tissue extracts, biological fluids



Application 1:

Identification of HIV Replication Inhibitor

1. CAF (CD8+ antiviral factor) though to be related to AIDS development

2. Determined the identity of CAF with SELDI techniques : alpha-defensin -1, -2 and -3

3. Demonstrated de novo discovery of biomarker and multimarker patterns, identification of drug candidates and determination of protein functions



Application 2:

Multimarker Clinical Assays for Cancer

1. Early detection of cancer – critical in effective cancer treatment

2. Cancer biomarker – massive protein expression profiling

3. High throughput assay for multimarker provided by SELDI array and multivariate software algorithms produced high sensitivity and specificity.



1. SELDIProteinChip for Alzheimer’s Disease

2. Wide rage of samples

Small sample amount

3. SELDI using antibody protein array : Ab against N-terminal sequence of target peptides (beta-amyloid)

4. Discovered candidate biomarkers, related inhibitors, & their functions and peptide expression levels

Application 3:

Biomarker and Drug Discovery Applications in Neurological Disorders

Biotech 2012 spring-2-protein_chips

Technology

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