1. Protein Arrays (Biosurfaces for Proteome Research)

2. Outline Protein Analysis – Introduction Why ? How ?
New Protein Analysis Tools
Protein Arrays
SELDI MS – Based ProteinChip®

3. Central Dogma of Life DNA mRNA Protein Protection of DNA
Amplification of genetic information
Efficient regulation of gene expression

4. Proteins
20 amino acids
30,000 – 34,000 genes
2,000,000 proteins

5. Transcription regulation Immune response Other vital cellular actions
Protein Functions
Signal transduction
Transcription regulation
Immune response
Other vital cellular actions

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. 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

8. Protein Microarray Part1
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.

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 substrates
Polyacrylamide or agarose gels
Glass
Nanowells
Proteins deposited on chip surface by robots
Benfey & Protopapas, 2005

11. Protein Attachment Diffusion
Protein suspended in random orientation, but presumably active
Adsorption/Absorption
Some proteins inactive
Covalent attachment
Affinity
Orientation of protein precisely controlled
Diffusion
Adsorption/
Absorption
Covalent
Affinity
Benfey & Protopapas, 2005

12. 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

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. 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.

17. Resources

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
, Vol 293, Science, 2001

20. “Global Analysis of Protein Activities Using Proteome Chips”
Snyder Lab, Yale University, , 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.

21. “Global Analysis of Protein Activities Using Proteome Chips”
Snyder Lab, Yale University, , 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.

22. “Global Analysis of Protein Activities Using Proteome Chips”
Snyder Lab, Yale University, , 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.

23. “Global Analysis of Protein Activities Using Proteome Chips”
Snyder Lab, Yale University, , 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.

24. “Global Analysis of Protein Activities Using Proteome Chips”
Snyder Lab, Yale University, , Vol 293, Science, 2001
Conclusions
1. Novel tool for protein interaction studies.
2. Concerns : * indirect interaction?
* missing proteins?
* surface chemistry?

25. Antibody Array for Protein Expression Profiling

26. SELDI MS-based ProteinChip
Part2
SELDI MS-based ProteinChip
Utilizes Surface Enhanced Laser Desorption/Ionization Mass Spectrometry (1993)
MALDI MS combined with chromatography (Bioaffinity): surface-MALDI

27. Protein Analysis by SELDI-MS1
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:

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

29. Mass Spectrometry

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
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
Benfey & Protopapas, 2005

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 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

35. Mass Spectrum of Protein mixture

36. Advantages of Mass Spectrometry
1. No labeling required.
2. Fast separation.
3. Multiplexing feasibility.
4. High sensitivity.

37. Disadvantages of Mass Spectrometry
1. Lower sensitivity compared to array.
2. Lower accuracy in quantitative assay.
3. Stringent sample purity.

38. “SELDIProteinChip Array Technology: Protein-Based Predictive Medicine and Drug Discovery Applications”
Ciphergen Biosystems, Inc, , Vol 4, J. Biomed. & Biotechnol., 2003

39. SELDIProteinChip Array Technology
“SELDIProteinChip Array Technology: Protein-Based Predictive Medicine and Drug Discovery Applications”
Ciphergen Biosystems, Inc, , 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

40. Identification of HIV Replication Inhibitor
“SELDIProteinChip Array Technology: Protein-Based Predictive Medicine and Drug Discovery Applications”
Ciphergen Biosystems, Inc, , 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. Multimarker Clinical Assays for Cancer
“SELDIProteinChip Array Technology: Protein-Based Predictive Medicine and Drug Discovery Applications”
Ciphergen Biosystems, Inc, , 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. Biomarker and Drug Discovery Applications in Neurological Disorders
“SELDIProteinChip Array Technology: Protein-Based Predictive Medicine and Drug Discovery Applications”
Ciphergen Biosystems, Inc, , 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 N-terminal sequence of target peptides (beta-amyloid)
4. Discovered candidate biomarkers, related inhibitors, & their functions and peptide expression levels