125:583Protein Arrays
(Biosurfaces for Proteome Research)
November 27, 2006Sobin Kim
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
SELDI MS protein chips
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
Benfey & Protopapas, 2005
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
Benfey & Protopapas, 2005
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.
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.
“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-3-streptavidin
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?
“Global Analysis of Protein Activities Using Proteome Chips”
Snyder Lab, Yale University, 2101-2105, Vol 293, Science, 2001
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
1
2
3
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
Benfey & Protopapas, 2005
Mass Analyzer
Benfey & Protopapas, 2005
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”
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
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