125:583 Protein Arrays (Biosurfaces for Proteome Research)

1. 125:583Protein Arrays (Biosurfaces for Proteome Research) November 27, 2006Sobin Kim

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

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

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

5. 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 • SELDI MS protein chips

8. Part1 Protein Microarray 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. Target: Proteins DNA (Big, 3D) (Small, 2D) Binding: 3D affinity 2D seq Stability: Low High Surface: Glass Glass Printing: Arrayer Arrayer Amplification: Cloning PCR Protein Array VS DNA Microarray

10. Protein substrates • Polyacrylamide or agarose gels • Glass • Nanowells • Proteins deposited on chip surface by robots Protein Array Fabrication Benfey & Protopapas, 2005

11. 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 Protein Attachment Adsorption/ Absorption Covalent Affinity Benfey & Protopapas, 2005

12. 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 Interactions 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. 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

17. “Global Analysis of Protein Activities Using Proteome Chips” Snyder Lab, Yale University 2101-2105, Vol 293, Science, 2001

18. “Global Analysis of Protein Activities Using Proteome Chips” Snyder Lab, Yale University, 2101-2105, 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.

19. “Global Analysis of Protein Activities Using Proteome Chips” Snyder Lab, Yale University, 2101-2105, 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.

20. “Global Analysis of Protein Activities Using Proteome Chips” Snyder Lab, Yale University, 2101-2105, 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.

21. “Global Analysis of Protein Activities Using Proteome Chips” Snyder Lab, Yale University, 2101-2105, 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.

22. “Global Analysis of Protein Activities Using Proteome Chips” Snyder Lab, Yale University, 2101-2105, Vol 293, Science, 2001 Conclusions 1. Novel tool for protein interaction studies. 2. Concerns : * indirect interaction? * missing proteins? * surface chemistry?

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

24. Protein Analysis by SELDI-MS 1 1) Apply sample (serum, tissue extract, etc.) to ProteinChip® array. 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 2 2) Wash sample with increasing stringency to remove non-specific proteins. 3 Source:http://dir.niehs.nih.gov/proteomics/emerg3.htm

25. 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

26. Mass Spectrometry

27. 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

28. 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 Ion Sources ESI MALDI Benfey & Protopapas, 2005

29. 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 Mass Analyzer Ion Trap Time of Flight Detector Benfey & Protopapas, 2005

30. 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.

31. 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 Protein Identification by MS

32. Mass Spectrum of Protein mixture

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

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

35. “SELDIProteinChip Array Technology: Protein-Based Predictive Medicine and Drug Discovery Applications” Ciphergen Biosystems, Inc, 237-241, Vol 4, J. Biomed. & Biotechnol., 2003

36. “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

37. “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

38. “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.

39. “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