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

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.

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.

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

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

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

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

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

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

“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 “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

Identification of HIV Replication Inhibitor “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

Multimarker Clinical Assays for Cancer “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.

Biomarker and Drug Discovery Applications in Neurological Disorders “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 N-terminal sequence of target peptides (beta-amyloid) 4. Discovered candidate biomarkers, related inhibitors, & their functions and peptide expression levels