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: Genechip DNA Microarray GC-LC / MS NMR Genomics Metabolomics Proteinchip Protein Array Proteomics

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

7. Proteinchip TM 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. Application of Crude Sample: Step:1 Proteinchip TM : Procedure

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

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

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

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

14. Protein Capture-chips – MS: Results

15. Proteinchip TM 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 Slide Preperation 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 Control Sample

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

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

23. Microarrayer : AFFIMATRICS GMS-417 Arrayer

24. Proteine Microarray:

26. Commercial Protein Measurement Technique:

27. 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.comwww.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