Transcript Protein arrays - Институт биоинформатики

Protein Arrays
(Biosurfaces for Proteome
Research)
Outline
Protein Analysis – Introduction
 Why ?
 How ?
New Protein Analysis Tools
 Protein Arrays
 SELDI MS – Based ProteinChip®
Central Dogma of Life
DNA
mRNA
 Protection of DNA
 Amplification of
genetic information
 Efficient regulation
of gene expression
Protein
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
Methods for Protein Analysis

Gel electrophoresis, northern/western
blot (fluorescence/radio active label)

X-ray crystallography

2D - mass spectrometry

Protein microarrays

Antibody Array for Protein Expression
Profiling
Part1
Protein Microarray
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.
Protein Array VS DNA Microarray
Target:
Binding:
Stability:
Surface:
Printing:
Amplification:
Proteins
(Big, 3D)
3D affinity
Low
Glass
Arrayer
Cloning
DNA
(Small, 2D)
2D seq
High
Glass
Arrayer
PCR
Protein Array Fabrication

Protein substrates




Polyacrylamide or
agarose gels
Glass
Nanowells
Proteins deposited
on chip surface by
robots
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
Benfey & Protopapas, 2005
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 function
Antigen–
antibody
Protein–
protein
Aptamers
Enzyme–
substrate
Receptor–
ligand
Benfey & Protopapas, 2005
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.
Detection

The preferred method of detection currently is
fluorescence detection. The fluorescent
detection method is compatible with standard
microarray scanners, the spots on the resulting
image can be quantified by commonly used
microarray quantification software packages.
However, some minor alterations to the analysis
software may be needed. Other common
detection methods include colorimetric
techniques based on silver-precipitation,
chemiluminescent and label free Surface
Plasmon Resonance.
Resources

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 University
2101-2105, Vol 293, Science, 2001
“Global Analysis of Protein Activities Using Proteome Chips”
Snyder Lab, Yale University, 2101-2105, 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.
“Global Analysis of Protein Activities Using Proteome Chips”
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.
“Global Analysis of Protein Activities Using Proteome Chips”
Snyder Lab, Yale University, 2101-2105, Vol 293, Science, 2001
Protein-Protein Interactions
1. Calmodulin-Biotin with Ca++.
2. Interaction checked with Cy-3streptavidin
3. Six calmodulin targets newly found.
4. Another six known targets could
not be detected.
“Global Analysis of Protein Activities Using Proteome Chips”
Snyder Lab, Yale University, 2101-2105, Vol 293, Science, 2001
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.
“Global Analysis of Protein Activities Using Proteome Chips”
Snyder Lab, Yale University, 2101-2105, Vol 293, Science, 2001
Conclusions
1. Novel tool for protein interaction
studies.
2. Concerns : * indirect interaction?
* missing proteins?
* surface chemistry?
Antibody Array for Protein
Expression Profiling

http://www.youtube.com/watch?v=EeiN6beb
CEw
Part2
SELDI MS-based ProteinChip

Utilizes Surface Enhanced Laser
Desorption/Ionization Mass Spectrometry
(1993)

MALDI MS combined with
chromatography (Bioaffinity): surfaceMALDI
Protein Analysis by SELDI-MS
1
1) Apply sample (serum,
tissue extract, etc.) to
ProteinChip® array.
2
2) Wash sample with increasing
stringency to remove non-specific
proteins.
3) Energy absorbing
molecules are added to
retained proteins.
Following laser desorption
and ionization of proteins,
Time-of Flight (TOF) mass
spectrometry accurately
determines their masses
3
Source:http://dir.niehs.nih.gov/proteomics/emerg3.htm
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)

ESI
MALDI
Extracts ions from sample
surface
Benfey & Protopapas, 2005
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-tocharge ratio
 Frequently used with
MALDI
Also other possibilities
Ion Trap
Time of Flight
Detector
Benfey & Protopapas, 2005
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 sample
 Extraction from a gel
 Digestion by proteases — e.g., trypsin

Mass spectrometer measures mass-charge ratio of
peptide fragments

Identified peptides are compared with database
 Software used to generate theoretical peptide
mass fingerprint (PMF) for all proteins in database
 Match of experimental readout to database PMF
allows 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”
Ciphergen Biosystems, Inc, 237-241, Vol 4, J. Biomed. & Biotechnol., 2003
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
“SELDIProteinChip Array Technology: Protein-Based Predictive Medicine and Drug
Discovery Applications”
Ciphergen Biosystems, Inc, 237-241, Vol 4, J. Biomed. & Biotechnol., 2003
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
“SELDIProteinChip Array Technology: Protein-Based Predictive Medicine and Drug
Discovery Applications”
Ciphergen Biosystems, Inc, 237-241, Vol 4, J. Biomed. & Biotechnol., 2003
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.
“SELDIProteinChip Array Technology: Protein-Based Predictive Medicine and Drug
Discovery Applications”
Ciphergen Biosystems, Inc, 237-241, Vol 4, J. Biomed. & Biotechnol., 2003
Application 3:
Biomarker and Drug Discovery
Applications in Neurological Disorders
1. SELDIProteinChip for Alzheimer’s Disease
2. Wide rage of samples
Small sample amount
3. SELDI using antibody protein array : Ab against Nterminal sequence of target peptides (beta-amyloid)
4. Discovered candidate biomarkers, related inhibitors, &
their functions and peptide expression levels