6. 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
7. Gel electrophoresis, northern/western
blot (fluorescence/radio active label)
X-ray crystallography
2D - mass spectrometry
Protein microarrays
Antibody Array for Protein ExpressionAntibody Array for Protein Expression
ProfilingProfiling
Methods for Protein AnalysisMethods for Protein Analysis
8. 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
9. Protein Array VS DNA Microarray
Target: Proteins DNA
(Big, 3D) (Small, 2D)
Binding: 3D affinity 2D seq
Stability: Low High
Surface: Glass Glass
Printing: Arrayer Arrayer
Amplification: Cloning PCR
10. Protein Array Fabrication
Protein substratesProtein substrates
Polyacrylamide orPolyacrylamide or
agarose gelsagarose gels
GlassGlass
NanowellsNanowells
Proteins depositedProteins deposited
on chip surface byon chip surface by
robotsrobots
Benfey & Protopapas, 2005
11. 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
12. 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 function
Receptor–
ligand
Antigen–
antibody
Protein–
protein
Aptamers
Enzyme–
substrate
13. Expression Array
Probes (antibody) on surface recognize
target proteins.
Identification of expressed proteins from
samples.
Typical quantification method for large # of
expressed proteins.
14. Interaction Array
Probes (proteins, peptides, lipids) on
surface interact with target proteins.
Identification of protein interactions.
High throughput discovery of interactions.
15. Functional Array
Probes (proteins) on surface react with
target molecules .
Reaction products are detected.
Main goal of proteomics.
16. DetectionDetection
The preferred method of detection currently isThe preferred method of detection currently is
fluorescencefluorescence detection. The fluorescentdetection. The fluorescent
detection method is compatible with standarddetection method is compatible with standard
microarray scanners, the spots on the resultingmicroarray scanners, the spots on the resulting
image can be quantified by commonly usedimage can be quantified by commonly used
microarray quantification software packages.microarray quantification software packages.
However, some minor alterations to the analysisHowever, some minor alterations to the analysis
software may be needed. Other commonsoftware may be needed. Other common
detection methods include colorimetricdetection methods include colorimetric
techniques based on silver-precipitation,techniques based on silver-precipitation,
chemiluminescent and label free Surfacechemiluminescent and label free Surface
Plasmon Resonance.Plasmon Resonance.
18. 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
19. “Global Analysis of Protein
Activities Using Proteome Chips”
Snyder Lab, Yale University
2101-2105, Vol 293, Science, 2001
20. 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
21. 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
22. 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
23. 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
24. 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
25. Antibody Array for ProteinAntibody Array for Protein
Expression ProfilingExpression Profiling
http://www.youtube.com/watch?v=EeiN6bebCEwhttp://www.youtube.com/watch?v=EeiN6bebCEw
26. SELDI MS-based ProteinChip
Utilizes Surface Enhanced Laser
Desorption/Ionization Mass Spectrometry
(1993)
MALDI MS combined with
chromatography (Bioaffinity): surface-
MALDI
Part2
27. 3) Energy absorbing3) Energy absorbing
molecules are added tomolecules are added to
retained proteins.retained proteins.
Following laser desorptionFollowing laser desorption
and ionization of proteins,and ionization of proteins,
Time-of Flight (TOF) massTime-of Flight (TOF) mass
spectrometry accuratelyspectrometry 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.) totissue extract, etc.) to
ProteinChip® array.ProteinChip® array.
2) Wash sample with increasing2) Wash sample with increasing
stringency to remove non-specificstringency to remove non-specific
proteins.proteins.
28. 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
30. 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
31. 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
32. 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
33. 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.
34. 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 ofMass 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 peptideSoftware 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 PMFMatch of experimental readout to database PMF
allows researchers to identify the proteinallows researchers to identify the protein
40. “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
41. “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.
42. “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