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High throughput Protein Measurement Techniques

High throughput Protein Measurement Techniques. Harin Kanani. Proteins:. Biological Macromolecules Functional Protein - Enzymes Structural Protein Antibody for Cell defense Signalling Proteins. Motivation for Protein measurement:. Basic biological research

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High throughput Protein Measurement Techniques

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  1. High throughput Protein Measurement Techniques Harin Kanani

  2. Proteins: • Biological Macromolecules • Functional Protein - Enzymes • Structural Protein • Antibody for Cell defense • Signalling Proteins

  3. Motivation for Protein measurement: • Basic biological research • Biomarkers : Molecular Classification & Diagnosis of disease • Drug Discovery • For profiling response of biological systems to toxins and pharmaceuticals

  4. Motivation High throughput Measurement: ::Genomic Revolution:: • Rapid (?) measurement of a large number of samples • non-hypothesis approach of research • Final Aim is - “Simultaneous, Complete, quantitative analysis of all Gene Expressions/ Proteins”

  5. High throughput Measurement: Proteomics Genomics • Proteinchip • Protein Array Metabolomics • Genechip • DNA Microarray • GC-LC / MS • NMR

  6. High throughput Measurement: Proteins Proteomics Genomics • Proteinchip • Protein Array Metabolomics • Genechip • DNA Microarray • GC-LC / MS • NMR

  7. ProteinchipTM Protein Capture Chips for Mass Spectrometry Using SELDI – Surface Enhanced Laser Disorption Ionization Or MALDI – Matrix Assisted Laser Disorption Ionization

  8. Protein Capture-chips:

  9. ProteinchipTM : Procedure Application of Crude Sample: Step:1

  10. ProteinchipTM : Procedure Wash chip to remove non-specifically binded protein/background: Step: 2

  11. Protein Capture-chips – MS: Procedure Add Energy Absorbing Molecules (EAS) or “Matrix”: Step: 3 This step loosens the protein binding

  12. Protein Capture-chips – MS: Procedure Use Laser for desorption and ionization of protein : Step: 4 TOF (Time of Flight Mass Spectrometer): Measure Molecular weight – Get Spectrum

  13. Protein Capture-chips – MS: Results Generating m/z vs Intensity Plots Step: 5

  14. Protein Capture-chips – MS: Results

  15. ProteinchipTM CIPHERGEN Proteinchip Biology System

  16. Protein Microarray High-throughput Quantification (Profiling) using specific antibody – antigen Interaction & relative intensity of floroscent signal

  17. Protein Microarray Technology:Slide Chemistry SlidePreperation Protein Imobilization Quenching Additional sites bovine serum albumin (BSA)

  18. Protein Microarray Technology:Protein Detection

  19. Protein Microarray Technology:Intensity vs. Protein Concentration

  20. Protein Microarray Expression Profiling Sample Control

  21. Protein Microarray Technology: Microarrayer Scanning Image Analysis Software Measure intensity with Cy-3 and Cy-5 Filter

  22. Protein Microarray Technology: Relative Protein Abundance In a sample Data Normalization Statistical Techniques To compare Relative Abundance in different samples Experimental Conclusion/ Discoveries

  23. Microarrayer : AFFIMATRICS GMS-417 Arrayer

  24. Proteine Microarray:

  25. Proteine Microarray:

  26. Commercial Protein Measurement Technique:

  27. Commercial Protein Measurement Technique: Many Techniques being explored… but very few Commercial products in the market Inspite of high demand Because……

  28. Protein Measurement: Bottlenecks.. • Capture agents: shortage of sufficiently specific antibodies or their ligands • Problem due to different forms of the post translational protein • Protein deactivation on micro array surfaces

  29. Protein Measurement: Bottlenecks.. • High throughput purification techniques which do not affect functional form of proteins • lack of uniform labelling due to different protein properties ..(upto 10 fold difference) • Varying protein concentration (~10^14) ..different concentration requires different conditions

  30. High throughput Protein Measurement Beginning of a long journey ahead….

  31. Thank You

  32. References: • Mitchell P, “ A perspective on protein microarray”, Nature Biotechnology, 20, 225 • Haab B., et. al., “ Protein microarray for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions”, Genome Biology 2001, 2(2):research/004.1 • Proteinchip Publications, literature and posters on: • www.ciphergen.com • MacBeath et al., “Printing Proteins as Microarrays for High-Throughput Function Determination”, SCIENCE, 289, 1760 • Zhu et. al., “ Global analysis of protein activities using proteome chips”, SCIENCE, 293, 2101

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