1. V. Protein Chips 1. What is Protein Chips 2. How to Make Protein Chips
3. MS-Based Protein Chip: SELDI

2. A protein microarray (or protein chip) is a high-throughput method used to determine the interactions, amounts and activities of proteins on a large scale. Large numbers of different molecules of protein have be arrayed on a support surface and then analyzed by a labeled probe or sample in parallel.

3. Motivation for Development of Protein Chips
Protein chips were developed due to the limitations of using DNA chips for determining gene expression levels in proteomics. The quantity of mRNA in the cell often doesn’t reflect the expression levels of the proteins they correspond to.
Additionally post-translational modifications, which are often critical for determining protein function, are not visible on DNA microarrays.

7. 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

8. 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

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. Expression Array
Probes (antibody) on surface recognize target proteins.
Identification of expressed proteins from samples.
Typical quantification method for large # of expressed proteins.

11. Interaction Array
Probes (proteins, peptides, lipids) on surface interact with target proteins.
Identification of protein interactions.
High throughput discovery of interactions.

12. Functional Array
Probes (proteins) on surface react with target molecules .
Reaction products are detected.
Main goal of proteomics.

13. 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

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

15. 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:

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

17. 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

18. 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

19. 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.

20. 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