Your SlideShare is downloading. ×
0
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Biotech 2012 spring-2-protein_chips
Upcoming SlideShare
Loading in...5
×

Thanks for flagging this SlideShare!

Oops! An error has occurred.

×
Saving this for later? Get the SlideShare app to save on your phone or tablet. Read anywhere, anytime – even offline.
Text the download link to your phone
Standard text messaging rates apply

Biotech 2012 spring-2-protein_chips

339

Published on

Published in: Technology
0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total Views
339
On Slideshare
0
From Embeds
0
Number of Embeds
1
Actions
Shares
0
Downloads
54
Comments
0
Likes
0
Embeds 0
No embeds

Report content
Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
No notes for slide

Transcript

  • 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. DNA mRNA Protein Central Dogma of Life 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. 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.  Gel electrophoresis, northern/western blot (fluorescence/radio active label)  X-ray crystallography  2D - mass spectrometry  Protein microarrays  Antibody Array for Protein ExpressionAntibody Array for Protein Expression ProfilingProfiling Methods for Protein AnalysisMethods for Protein Analysis
  • 8. 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
  • 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 substratesProtein substrates  Polyacrylamide orPolyacrylamide or agarose gelsagarose gels  GlassGlass  NanowellsNanowells  Proteins depositedProteins deposited on chip surface byon chip surface by robotsrobots Benfey & Protopapas, 2005
  • 11. 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
  • 12. 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
  • 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. DetectionDetection  The preferred method of detection currently isThe preferred method of detection currently is fluorescencefluorescence detection. The fluorescentdetection. The fluorescent detection method is compatible with standarddetection method is compatible with standard microarray scanners, the spots on the resultingmicroarray scanners, the spots on the resulting image can be quantified by commonly usedimage can be quantified by commonly used microarray quantification software packages.microarray quantification software packages. However, some minor alterations to the analysisHowever, some minor alterations to the analysis software may be needed. Other commonsoftware may be needed. Other common detection methods include colorimetricdetection methods include colorimetric techniques based on silver-precipitation,techniques based on silver-precipitation, chemiluminescent and label free Surfacechemiluminescent and label free Surface Plasmon Resonance.Plasmon Resonance.
  • 17. ResourcesResources  http://www.microarraystation.com/http://www.microarraystation.com/
  • 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 2101-2105, Vol 293, Science, 2001
  • 20. 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, 2101-2105, Vol 293, Science, 2001
  • 21. 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, 2101-2105, Vol 293, Science, 2001
  • 22. 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. “Global Analysis of Protein Activities Using Proteome Chips” Snyder Lab, Yale University, 2101-2105, Vol 293, Science, 2001
  • 23. 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, 2101-2105, Vol 293, Science, 2001
  • 24. 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, 2101-2105, Vol 293, Science, 2001
  • 25. Antibody Array for ProteinAntibody Array for Protein Expression ProfilingExpression Profiling  http://www.youtube.com/watch?v=EeiN6bebCEwhttp://www.youtube.com/watch?v=EeiN6bebCEw
  • 26. SELDI MS-based ProteinChip  Utilizes Surface Enhanced Laser Desorption/Ionization Mass Spectrometry (1993)  MALDI MS combined with chromatography (Bioaffinity): surface- MALDI Part2
  • 27. 3) Energy absorbing3) Energy absorbing molecules are added tomolecules are added to retained proteins.retained proteins. Following laser desorptionFollowing laser desorption and ionization of proteins,and ionization of proteins, Time-of Flight (TOF) massTime-of Flight (TOF) mass spectrometry accuratelyspectrometry accurately determines their massesdetermines their masses Protein Analysis by SELDI-MS Source:http://dir.niehs.nih.gov/proteomics/emerg3.htm 1 2 3 1) Apply sample (serum,1) Apply sample (serum, tissue extract, etc.) totissue extract, etc.) to ProteinChip® array.ProteinChip® array. 2) Wash sample with increasing2) Wash sample with increasing stringency to remove non-specificstringency to remove non-specific proteins.proteins.
  • 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 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
  • 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 samplePreparation of protein sample  Extraction from a gelExtraction from a gel  Digestion by proteases — e.g., trypsinDigestion by proteases — e.g., trypsin  Mass spectrometer measures mass-charge ratio ofMass spectrometer measures mass-charge ratio of peptide fragmentspeptide fragments  Identified peptides are compared with databaseIdentified peptides are compared with database  Software used to generate theoretical peptideSoftware used to generate theoretical peptide mass fingerprint (PMF) for all proteins in databasemass fingerprint (PMF) for all proteins in database  Match of experimental readout to database PMFMatch of experimental readout to database PMF allows researchers to identify the proteinallows 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, 237-241, Vol 4, J. Biomed. & Biotechnol., 2003
  • 39. “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
  • 40. “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
  • 41. “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.
  • 42. “SELDIProteinChip Array Technology: Protein-Based Predictive Medicine and Drug Discovery Applications” Ciphergen Biosystems, Inc, 237-241, Vol 4, J. Biomed. & Biotechnol., 2003 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 Application 3: Biomarker and Drug Discovery Applications in Neurological Disorders

×