125:583 Protein Arrays (Biosurfaces for Proteome Research) November 27, 2006 Sobin 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 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: ProteinsDNA (Big, 3D)(Small, 2D) Binding: 3D affinity2D seq Stability: LowHigh Surface: GlassGlass Printing: ArrayerArrayer Amplification: CloningPCR

Protein Array Fabrication Protein substrates Protein substrates Polyacrylamide or agarose gels Polyacrylamide or agarose gels Glass Glass Nanowells Nanowells Proteins deposited on chip surface by robots Proteins deposited on chip surface by robots 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 function Receptor– 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 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. “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. “Global Analysis of Protein Activities Using Proteome Chips” Snyder Lab, Yale University, , Vol 293, Science, 2001

Protein-Protein Interactions 1. Calmodulin-Biotin with Ca 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, , 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, , 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, , 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 molecules are added to retained proteins. Following laser desorption and ionization of proteins, Time-of Flight (TOF) mass spectrometry accurately determines their masses Protein Analysis by SELDI-MS Source: ) Apply sample (serum, tissue extract, etc.) to ProteinChip® array. 2) Wash sample with increasing stringency to remove non-specific 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 sample Preparation of protein sample Extraction from a gel Extraction from a gel Digestion by proteases — e.g., trypsin Digestion by proteases — e.g., trypsin Mass spectrometer measures mass-charge ratio of peptide fragments Mass spectrometer measures mass-charge ratio of peptide fragments Identified peptides are compared with database Identified peptides are compared with database Software used to generate theoretical peptide mass fingerprint (PMF) for all proteins in 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 Match of experimental readout to database PMF allows 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, , Vol 4, J. Biomed. & Biotechnol., 2003

“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

“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 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, , 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, , Vol 4, J. Biomed. & Biotechnol., 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