Protein microarrays, ICAT, and HPLC protein purification


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  • EXTRA NOTES: Reservoir holds the solvent. Usually min 2 reservoirs, each with up to 1,000 cc of solvent. Each reservoir usually fitted with diffuser /degasser through which He is bubbled, dissolve gases from mobile phases Pump generates the specific flow of the mobile phase Sample injection loop : can be manual and/or automated. Autosampler introduces the solvent into phase stream that carries it into the high pressure column Column contains the stationary phase where separation occurs Detector sees the separation bands as they elute out of the column Detector sends info to computer that generates and stores the chromatogram Mobile phase exits detector and is sent to waste or collected.
  • Protein microarrays, ICAT, and HPLC protein purification

    1. 1. • ICAT (Isotope-Coded Affinity Tag) • Protein Purification with HPLC • Protein MicroarraysRaul Soto Biol502 Fall 2011
    2. 2. Isotope-Coded Affinity Tag (ICAT)Raul Soto Biol502 Fall 2011
    3. 3.  Identification and quantification of complex protein mixtures (quantitative proteomics) Uses chemical labeling reagents that specifically label a Cys residues in vitro method Gel – free
    4. 4. Biotin Affinity tag: enables Thiol-Specific Reactive group: bindsthe isolation of the covalently and labels Cysteine residuespeptide/protein by affinitychromatography O Linker: Heavy version will have deuteriums at *NH Light version will have hydrogens at * NH H H N * O O * N * I * O O S O
    5. 5. 4 Main Steps: 1 Gygi S, et al (1999)1 Lyse and Label: Side chains of Cys1. residues in reduced protein sample for one cell state are tagged with light ICAT. 2 Equivalent groups in protein sample for the second cell state are tagged with the heavy ICAT. 3 42 Proteolysis: The two samples are2. combined and enzymatically cleaved to produce peptide fragments. Some of these fragments will be tagged3 Affinity isolation: Tagged, Cys-3. containing fragments are isolated using avidin chromatography4 ID and quantification: Isolated peptides4. are separated and analyzed using LC- MS/MS
    6. 6. 1 Lyse and Label Label sample 1 (cell state 1) with isotopically light probe (d0) Label sample 2 (cell state 2) with isotopically heavy probe (d8)
    7. 7. 2 Proteolysis Mix the samples together Cleave enzymatically to generate peptide fragments. Some fragments will contain the ICAT tag
    8. 8. 3 Affinity isolation Subject both samples to avidin affinity chromatography  Isolates fragments labeled with isotope-coded tags  Uses specific interaction between immobilized avidin on the column and biotin on the linker
    9. 9. Biotin Affinity tag: enables Thiol-Specific Reactive group: bindsthe isolation of the covalently and labels Cysteine residuespeptide/protein by affinitychromatography O Linker: Heavy version will have deuteriums at *NH Light version will have hydrogens at * NH H H N * O O * N * I * O O S O
    10. 10. ID and quantification4 LC-MS/MS (Liquid chromatography – tandem mass spectrometry):  Determines quantity and sequence identity of proteins from which the tagged peptides were obtained  Technique can be used for complex protein mixture
    11. 11. ICAT Helps you answer questions such as: Which proteins are being expressed in Experimental vs Control? Which proteins are up-regulated or down-regulated in Experimental vs Control? How much are proteins A, B, C being up/down- regulated?
    12. 12. Advantages Disadvantages Accurate: Relative protein  Bias for Cys-rich proteinslevels between samples can beestimated within 10% accuracy  Large ICAT reagent interferes with MS fragmentation Can be used on complex  New: Cleavable reagent that canmixtures of proteins be removed after separation, before MS Highly automated  Tag size reduces the quality of Peptides sequenced directly MS datausing MS/MS  Expensive
    13. 13. Example (Gygi, 1999) Compared protein expression in yeast S. cerevisiae, using ethanol or galactose as carbon source Detected differences in protein expression were consistent with known metabolic function in yeast under glucose-repressed conditions. Gygi S, et al (1999)
    14. 14. Example (Gygi, 1999) Gygi S, et al (1999)
    15. 15. Protein Purification using HPLCsRaul Soto Biol502 Fall 2011
    16. 16. What is it? Process to isolate a single type of protein from a complex mixtureWhy? Research: Purified protein necessary to characterize the function, structure, and interactions of a protein of interest Drug Development : Protein-based drug formulation development Drug Manufacturing: at significantly larger volumesHow? Using differences in protein size, charge, physical and chemical properties, binding affinity, biological activity.
    17. 17.  Flasks 5 mL  Spinner flasks (roller bottles) 50-200mL  Bench Top Bioreactors 5-10 L  Pilot Scale Bioreactors 50-200L  Production Vessels 20,000 L
    18. 18. Protein molecules in a solution (mobile phase) areseparated based on differences in chemical or Cphysical interaction with a stationary material (solidphase). O Gel filtration (size) L Size-exclusion (size) U Ion-exchange (charge) Affinity chromatography : (binding affinity) M  Receptor / ligand N  Enzyme / substrate  Immunoaffinity : antigen / antibody  Metal Binding : covalent bond of residues (esp. histidine) to metals (Ni2+, Cu2+, Zn2+, Bi3+)
    19. 19.  STEP 1 : prepare solid phase: a solid matrix with a ligand coupled (usually covalently) Matrix: agarose, sephadex, cellulose, other polymers) STEP 2: pass mobile phase through the solid phase the target protein will bind noncovalently to the ligand molecules in the solid phase’s resin matrix. The rest of the mobile phase elutes out of http://www.molecular- the colum
    20. 20.  STEP 3 : Unbind protein of interest from the solid phase. An elution buffer disrupts protein-ligand interaction (pH extremes, high salt, detergents, chaotrophic agents, etc.) Denaturing agents like urea can also break protein-ligand interaction by changing the configuration of the protein active site. A single pass through an affinity column can achieve 1,000 – 10,000 fold purification of a ligand from a mixture hrom.jpg
    21. 21.  HPLC : High performance/pressure liquid chromatography A highly improved form of column chromatography Substance is forced to elute through the column under high pressures (up to 400 bar in HPLC, up to 6,800 bar in Ultra HPLC). FPLC : Fast Protein Liquid Chromatography Standard working pressure is “only” 3 – 5 MPa (approx 30 – 50 bar) /products/Agilent- HP_1100_Series_HPLC_MWD_System.JPG
    22. 22.  Advantages of using HPLCs over regular column chromatography:  Higher throughput  Faster  Can use much smaller particle size for column packing material, which means greater surface area for interactions between mobile and solid phases  Highly automated, can use extremely sensitive detection methods  Autosampler: HPLC can run several samples in order
    23. 23.  text HPLC Apparatus Overview1. Solvent reservoirs 7. Sample injection loop2. Solvent degasser 8. Pre-column (guard column)3. Gradient valve 9. Analytical column4. Mixing vessel for delivery of the mobile 10. Detector (IR, UV) phase 11. Data acquisition (PC)5. High pressure pump 12. Waste or fraction collector6. Switching valve in “inject” position6’. Switching valve in “load” position
    24. 24. Bottle tray kits Solvent reservoirs PC: processing, display, file C Pre-heaters (LIMS, CDS) O Heaters / Coolers L Multiple columns Raw data U Auto-switching Pump M N Detector Sample Purified protein injectorAutosamplersHeaters / coolers • Mass Spec • sample storage
    25. 25.  Different peaks correspond to different components in the mixture Y axis – absorbance X axis – time, % concentration, flow rate Qualitative assessments: compare peak positions vs standard Quantitative assessments: assessment of relative concentrations of components can be obtained from peak area comparisons Column performance: indicated by comparison against standards
    26. 26.  Normal Phase:  Reversed Phase (most common):  Hydrophilic silica particles in solid  Hydrophobic hydrocarbon chains phase in solid phase  Hydrophobic solvent  Hydrophilic solvent  Hydrophilic molecules in mobile  Hydrophobic molecules in the phase bind to solid phase mobile phase form van der Waals  Hydrophobic molecules flow out bonds with hydrophobic first hydrocarbons in solid phase  Elution: from most to least  Hydrophilic molecules in the hydrophobic. mobile phase interact with hydrophilic solvent and flow out first  Elution: from least to most hydrophobic
    27. 27. Process Step Column Process Step down Flow direction Equilibration (equilibration / wash 1 buffer) Column up Flow direction Regeneration (regeneration buffer) down Load (harvest filtrate w/ protein) down Wash 1 Blank Elution4 – 6 cycles (equilibration/ wash 1 buffer) Sequence down (after last cycle, if required) STORAGE down Wash 2 EQUILIBRIUM (wash 2 buffer) down Elution down (elution buffer) Column Storage (storage buffer) down Cleaning (cleaning solution)
    28. 28.
    29. 29. Protein MicroarraysRaul Soto Biol502 Fall 2011
    30. 30.  A high density array containing 100’s – 1,000’s of proteins positioned in an addressable format Proteins or peptides are individually purified and arrayed on a surface (i.e. glass slide) Can screen multiple proteins simultaneously 2 main types: Functional and Analytical
    31. 31.  Diffusion  Protein suspended in Diffusion random orientation, but presumably active Adsorption/ Absorption Adsorption/Absorption  Some proteins inactive Covalent Covalent attachment  Some proteins inactive Affinity Affinity  Orientation of protein precisely controlled
    32. 32. Antigen–  Different capture molecules antibody must be used to study different interactions Protein– protein  Examples  Antibodies (or antigens) for detection Aptamers  Proteins for protein-protein Enzyme– interaction substrate  Enzyme-substrate for biochemical function Receptor– ligandAptamer = single-stranded short oligonucleotides
    33. 33.
    34. 34.  Can be used to identify substrates of enzymes of interest  Identify protein targets of biologically active small molecules (drug and drug target ID)  Protein – protein interactions  Post-translational modifications  Useful for rapid analysis of proteomes and other large collections of proteins
    35. 35.  Different types of ligands (antibodies, antigens, nucleic acid aptamers, carbohydrates, small molecules) with high affinity and specificity, are arrayed onto a surface  Quantify levels of different proteins by binding exact proteins to microarray  Monitoring protein expression levels, protein profiling  Clinical diagnostics  Similar to DNA arrays, protein samples from two biological states can be compared by labeling with red and green fluorescent dyesAptamer = single-stranded short oligonucleotides
    36. 36. Characteristic Protein MA DNA MATarget Large 3D molecule Smaller 2D moleculeBinding 3D affinity 2D sequenceStability Low HighAmplification Cloning PCRBiochemical Behavior Proteins behave in DNA will behave similarly diverse and unique under single hybridization ways conditionCost $500-$1000 per $10 per oligo antibody
    37. 37.  Non-specific binding:  Adjust solute conditions: salt concentration, pH, etc Protein on array surface not in the right conformation  The protein should be folded, not denatured  The protein orientation should be correct (anchored by the same aminoacid)  The protein should be kept away from the surface by a linker, to avoid steric hindrance Denaturation  Solution conditions  Different proteins like different conditions, no binding may be caused by inappropriate conditions (pH, temperature, etc.)  Variability in results may be reduced by using an efficient lysis buffer, maintaining consistent sample processing conditions
    38. 38. References
    39. 39.  [1] Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R (October 1999). "Quantitative analysis of complex protein mixtures using isotope-coded affinity tags".Nature Biotechnology 1999. 17 (10): 994-9. PMID 10504701. [2] Isotope-coded affinity tags (ICATs) – Wikipedia  Accessed 03 Dec 2011. [3] Project OSCAR : Quantitative Proteomics – ICAT  %20ICAT/Quantitative%20Proteomics%20-%20ICAT/Contents/print.pdf Accessed 03 Dec 2011. [4] Sethuraman M, et al. Isotope-coded Affinity Tag Approach to Identify and Quantify Oxidant-sensitive Protein Thiols. Molecular & Cellular Proteomics 2004. 3:273-278. PMID: 14726493. [5] Sethuraman M, et al. Isotope-coded Affinity Tag (ICAT) Approach to Redox Proteomics: Identification and Quantitation of Oxidant-Sensitive Cysteine Thiols in Complex Protein Mixtures. Journal of Promeome Research. 2004. 3: 1228-1233. PMID: 15595732. [6] Turecek, J. Mass spectrometry in coupling with affinity capture-release and isotope-coded affinity tags for quantitative protein analysis. Mass Spectrom. 2002, 37, 1-14. PMID 11813306. [7] US 6670194 "Rapid quantitative analysis of proteins or protein function in complex mixtures," Rudolf Hans Aebersold et al. (PATENT)  Accessed 03 Dec 2011.
    40. 40.  [1] Affinity Chromatography, University College London  Accessed 03 Dec 11 [2] Chromatography: The Chromatogram, Rensselaer Polytechnic Institute  Accessed 03 Dec 11 [3] Chromatography – Wikipedia  Accessed 03 Dec 11 [4] High Performance Liquid Chromatography HPLC, Chemguide  Accessed 03 Dec 11 [5] High Performance Liquid Chromatography – Wikipedia  Accessed 03 Dec 11 [6] Protein Purification – Wikipedia  Accessed 03 Dec 11 [7] Protein Purification in One Day  Accessed 03 Dec 11 [8] The Protein Purification Facility, Hebrew University of Jerusalem  Accessed 03 Dec 11 [9] Thermo Scientific Pierce Protein Purification Technical Handbook  Accessed 03 Dec 11
    41. 41.  [1] Antibody Microarray – Wikipedia.  Accessed 03 Dec 11 [2] Chandra H, Reddy PJ, and Srivastava S. Protein Microarrays and Novel Detection Platforms. Expert Rev Proteomics. 2011; 8:61-79. PMID: 21329428 [3] Fasolo J, Snyder M. Protein Microarrays. Methods in Molecular Biology. 2009; 548:290-222. PMID: 19521827 [4] Merbl Y, Kirschner M. Protein Microarrays for Genome-Wide PostTranslational Modification Analysis. Wiley Interdiscip Rev Syst Biol Med. 2011; 3(3): 347-56. PMID : 20865779 [5] Phizicky E, Bastiaens PIH, Zhu H, Snyder M and Fields S. Protein Analysis on a Proteomic Scale. Nature. 2003; 422:208-205. PMID: 12634794 [6] Protein Microarray – Wikipedia.  Accessed 03 Dec 11 [7] Talapatra A, Rouse R and Hardiman G. Protein Microarrays: Challenges and Promises. Pharmacogenomics. 2002; 3(4). PMID: 12164775 [8] Zhu H and Snyder M. Protein Chip Technology. Current Opinion in Chemical Biology. 2003. 7:55-63. PMID: 12547427
    42. 42. Questions ?
    43. 43. Extra Slides
    44. 44.  “… to define the identities, quantities, structures, and functions of complete complements of proteins, and to characterize how these properties vary in different cellular contexts.” (Phizicky 2003)
    45. 45.  More complex than genomics  Organism’s genome remains more or less constant  Proteome changes depending on  Cell type  Tissue  Organ  Development stage (embryo, fetus, child, adult)  Dynamic responses to environmental signals  Disease state  Gene activity  Post-translational modifications
    46. 46.  Time it takes a particular compound to travel through the column Depends on:  Pressure used  Temperature of column  Nature of stationary phase  Solvent composition You must control all these conditions carefully if you are using retention time as a way to identify compounds.