Biochem Ch 8 & 9

  1. Reasons for protein purification
    • Scientific reasons- to study protein function
    • Therapeutic purposes- i.e production of insulin
    • Use of enzymes in pharmaceutics and industry
  2. Old way of protein purification
    • Identify an organism/organ/tissue that produces a protein of interest in "large" quantities
    • Homogenize the tissue and extract your protein together with thousands of others
    • Perform numerous selective separation steps until the protein is isolated to sufficient purity
  3. Protein purification now
    • Identify a gene of interest, isolate DNA, amplify by PCR, clone to a plasmid
    • Over-express the protein in an appropriate organism: bacterial cells, yeast, insect
    • Break open the cells to extract protein and perform selective separation steps until the protein is isolated to sufficient purity
  4. What properties to consider when separating proteins
    • Solubility 
    • Ionic charge
    • Polarity/hydrophobicity
    • Size
    • Binding specificity
  5. Gel eletrophoresis
    • Molecules with a net charge will migrate in an electric field with a velocity proportional to:
    • Charge density, size and shape

    Generally used as an analytical technique, occasionally as a preparative technique
  6. Zonal electrophoresis
    Constrain ions to move within a solid support (such as a thin gel between glass plates) to minimize diffusion
  7. PAGE: Polyacrylamide gel electrophoresis
    • Top and bottom buffer reservoirs are connected by a porous gel
    • Top reservoir is typically connected to the cathode; the bottom to the anode
  8. How do we ensure that all proteins run in the same direction?
    The electric field; the two chambers are only connected with the gel so protein must go through
  9. Image Upload 1
    • Acrylamide and methylenebisacrylamide
    • Used in PAGE
  10. How does acrylamide and methylenebisacrylamide work in PAGE
    Image Upload 2
  11. Mesh density is controlled by what in PAGE
    • Concentration of acrylamide
    • Degree of crosslinking (concentration of methylenebisacrylamide)
  12. How does PAGE work?
    • The mixture of macromolecules will go in porous gel
    • Once you run the electrophoresis then the large proteins tangle in the gel and have slower migration
  13. Density of the cross-linked gel determines the size range for proteins that move well through gel. What does high density gels separate? low density? Gradient of density?
    • High: better separation of small proteins
    • Low: large proteins
    • Gradient of density: both large and small
  14. SDS-Page
    • Denaturing gel electrophoresis
    • SDS: detergent with C12 hydrocarbon chain (hydrophobic) and a negatively charged sulfate (highly polar)
    • SDS bonds to hydrophobic regions of protein side chains
  15. How does SDS-PAGE work?
    • SDS denatures protein tertiary structure by disrupting internal charge and hydrophobic interactions -> uniform shape
    • Overwhelms protein native charge -> uniform negative charge
    • All proteins now have a uniform charge/mas ration
  16. Result of SDS-PAGE
    Mobility of proteins towards the positive electrode (anode) depends only on their size
  17. Components of the sample buffer in SDS-PAGE
    • SDS: denaturing detergent; unfolds and coats proteins with negative charge
    • Reducing agents (beta-mercaptoethanol; dithiothreitol, TCEP): break any disulfide bonds and prevent random formation of bonds between different proteins
    • Blue dye: to visualize the solvent front
    • Glycerol: increases density of sample, so it drops to the bottom of the sample well buffer to maintain pH
    • Boil for 3-5 minutes to ensure maximal unfolding
  18. Reduction in SDS-PAGE
    Reduction of covalent protein disulfide bondsImage Upload 3
  19. Common reducing agents:
    Image Upload 4
  20. SDS-PAGE: Determining Molecular Weight
    • Using standard proteins with known molecular weights (MW) to calibrate
    • Then measure and plot distance migrated of protein standards vs. log of their MW
    • Draw a standard curve through the linear range and measure the distance migrated of your unknown
    • Locate the corresponding log MW and find the MW of the unknown protein

    Image Upload 5
  21. How does coomassie brilliant blue dye affect protein visualization?
    Complexes with the unfolded proteins (detects ~50 ng)
  22. How does silver stain affect SDS-PAGE?
    Improves sensitivity by ~50 times to ~1 ng
  23. Western blotting
    • Detect a specific protein by staining with antibody 
    • Sensitivity depends on antibody, but can be way below 1 ng
  24. Denaturing electrophoresis
    • SDS-PAGE typical example
    • Denature proteins with SDS and heating, break up complexes, break down disulfide bonds, then run gel
    • Use to show what proteins are present in a mixture
  25. Native electrophoresis
    • Run electrophoresis without SDS/head denaturation to maintain folded proteins/complexes/associations
    • Due to difference in charge, proteins might run in opposite directions or not at all
    • Often is used to demonstrate interactions (protein-protein, protein-nucleic acid interactions)
    • Can be reducing or non-reducing
  26. SDS-PAGE vs native PAGE
    Image Upload 6
  27. Isoelectric Point (pI)
    • The pH at which the protein carries no net charge
    • Technically, pI= 1/2(pK1+pK2) for transitions around neutral
  28. When are proteins negatively charged vs positively charged?
    • When pH > pI protein is negatively charged
    • When pH < pI protein will be positively charged
  29. pH=pI
    The minimal solubility of a folded protein come from this because of interactions with water as a solvent
  30. What is the pI for a neutral amino acid?
    • Will be equal to the average between the pKa values of ionizable groups
    • Image Upload 7
  31. When is a proteins calculated pI no longer accurate?
    When it is beyond ~1 pH unit
  32. How can you estimate the pI of a protein?
    • If a protein contains 4 Arg, 10 Glu, 3 Asp, 2 Lys and no other charged AAs
    • it will have 6 positive charges, 13 negative charges
  33. Isoelectric focusing (IEF)
    • Electrophoresis in a pH gradient
    • Molecules run in a pH gradient until they reach the pH equal to their pI
  34. What can isoelectric focusing be used for?
    • Experimental determination of pI of a peptide or protein
    • To separate proteins/peptides based on the difference in their pI values
  35. Function of IEF
    • At a very low pH, all molecules are positively charged
    • As (+)ve ions move towards the (-)ve pole, the pH increases and they become less protonated until at their pI, the net charge is 0
    • As (-)ve ions move towards the (+)ve pole, the pH decreases and they become more protonated until at their pI
  36. What solution is incorporated into a gel?
    Image Upload 8
  37. 2D electrophoresis
    • Used to: 
    • Separate complex protein mixtures
    • Detect posttranslational modifications (PTM) i.e phosphorylation can strongly affect protein pI and therefore can be easily detected
    • 1st dimension-IEF
    • 2nd dimension-PAGE
  38. Column Chromatography
    Powerful methods for protein purification and characterization based on differential interaction of proteins (in mobile phase) with a solid column matrix (stationary phase)
  39. How does column chromatography work?
    Uses interactions between a protein mixture (mobile phase) and a solid support (stationary phase) for separation based on a single physical property including size, charge, hydrophobicity, specific interaction
  40. Elution
    The process of flowing the protein out of the column
  41. Types of chromatography
    • Stationary phase (column, matrix, beads):
    • Ion exchange
    • Gel filtration
    • Reverse-phase
    • Affinity

    Mobile phase (solvent, conditions required to elute protein) depends of protein and column used
  42. Ion exchange chromatography
    • Electrostatic interactions between a charged protein (above or below the pI) and a charged stationary phase
    • A positively charged protein (basic) will bind stronger to a negatively charged matrix (acidic matrix)
    • A negatively charged protein will bind stronger to a positively charged matrix (basic matrix)
  43. Gel filtration
    • Allows for the separation of proteins based on size and shape (also known as size exclusion chromatography)
    • Solid phase: beads with pores of a defined shape and size
    • larger proteins pass through a smaller volume (only between the beads) where smaller proteins can get into beads which makes for separation and a longer path
    • Different resins target different weight
  44. Reverse-phase
    Hydrophobic stationary phase (hydrophobicity-based separation)
  45. Advantages of engineered affinity tags
    • Unrelated to target protein
    • Sizes range from a few AA's to entire fusion proteins
    • Fusion to N-terminal or C-terminal end of target protein
  46. Polyhistidine
    • 6-10 His in a row bind tightly to nickel beads
    • Eluted by addition of imidazole (looks like His side chain)
  47. MBP
    • Maltose binding protein binds to amylose (linear chain of glucose) resin
    • Eluted by addition of maltose (glucose-based disaccharide)
  48. GST
    • The enzyme glutathione-S-Transferase (GST) binds tightly to glutathione (a tripeptide)
    • Eluted by addition of excess of free glutathione
  49. Affinity
    • Separation base on specific interactions (i.e enzyme-substrate; protein-ligand; protein-metal)
    • Unwanted proteins are washed through the column and protein of interest is eluted by ligand solution
    • Kids have "higher affinity" to playgrounds than adults do, and can be separated from adults by passing them through playgrounds. Then, kids can be eluted from the solid matrix by passing other kids who have not played yet.
  50. Anion exchange
    Positively charged (basic) matrix that interacts with anions
  51. Cation exchange
    Negatively charged (acidic) matrix that interacts with cations
  52. How does Ion exchange chromatography work?
    • Choose pH where protein has an appropriate charge
    • Proteins will bind with beads through electrostatics
    • Salt (Na or CL) will compete for interactions and exchange the ions
    • Use salt gradient to promote release (elution) of the bound proteins
    • Image Upload 9
  53. IEC elution by salt gradient
    • A concentration gradient of salt causes fractionation, and separation of multiple protein species based on strength of interaction (red>>purple>>yellow at a particular pH)
    • High concentration of salt will elute all proteins from the column
    • Image Upload 10
  54. When is gel filtration useful?
    • To separate proteins with at least 2x size ration
    • Can separate 15kDa from 30kDa
    • Probably not 30kDa from 45kDa
  55. Protein Dialysis
    • Another size-based approach
    • Removing small molecules (salts, buffer, etc) by diffusion via semi-permeable membrane with small pores while retaining proteins inside the dialysis bag
  56. Amino acid analysis
    Provides information about content, not the sequence
  57. How to determine a protein sequence?
    • How many chains (quaternary structure)?
    • Break protein down into short peptides
    • Analyze peptide sequences
    • Combine all of these into a primary sequence
    • Special consideration: disulfide bonds
  58. Fragmenting proteins
    • Proteins are too long to be sequenced at once
    • Peptide bonds can be specifically cleaved by peptidases/proteases
  59. Peptidase
    Breaks peptide bond
  60. Endopeptidase
    Breaks peptide bond somewhere in the middle of (inside) the sequence
  61. Exopeptidase
    Breaks peptide bond on a terminal amino acid ("exterior" peptide bond)
  62. Carboxypeptidase
    Exopeptidase that hydrolyzes peptide bond at the C-terminus (carboxyl terminus)
  63. Aminopeptidase
    Exopeptidase that hydrolyzes peptide bond of the N-terminal residue (amino terminus)
  64. Image Upload 11
    • Carboxymethylation
    • Cys irreversible modification is required to prevent oxidation
  65. Image Upload 12
    • Reducing disulfide bonds
    • Using DTT Cleland's Reagent
    • Image Upload 13
  66. Trypsin
    • Basic residues
    • Cleavage points: lys, arg
  67. Chymotrypsin
    • Aromatic residues
    • Cleavage points: phe, trp, tyr
  68. Staphylococcus aureus V8 protease
    Cleavage points: asp, glu
  69. Asp-N-protease
    Cleavage points: ASP, GLu
  70. Endo Lys C
    Lys (C)
  71. Cyanogen bromide
    • Chemical cleavage
    • Cleavage points: met (C)
  72. Overlapping peptide sequences
    • Make it possible to determine the original sequence of the polypeptide chain
    • Combine overlapping sequences together
  73. Edman degradation
    • Image Upload 14
    • Phenylisothiocyanate (PITC) is the reagentq
  74. Mass spectrometer common organization
    • Ion source, Mass analyzer, detector
    • All separation is based on mass to charge ratio
  75. Electrospray ionization (ESI)
    • Protein is supplied in a volatile buffer (i.e ammonium acetate)
    • Evaporation of a drop in vacuum results in charge repulsion and dispersion to smaller drops
    • Generates population of protein ions with many charge states
  76. ESI-MS
    • Mass reconstruction/deconvolution
    • Image Upload 15
  77. MALDI (matrix assisted laser desorption ionization) -- Matrix qualities
    • Peptides are mixed with matrix:
    • absorbs laser energy
    • often acids (source of H+)
    • small size to not interfere with peptides
    • Not volatile in vacuum
    • Relatively polar to dissolve in water solutions
  78. MALDI
    • Ionized biomolecules are separated in electric field
    • Mass can be determined from time it takes to reach the detector: time of flight (mass is directly proportional to time
    • Fewer charge states
  79. ESI vs MALDI
    • Electrospray MS: syringe full of protein/peptide solution
    • Ionize by concentrating charges in vacuum
    • Highly charged spectrum
    • Often connected to liquid chromatography systems
    • Can be used to analyze "native" proteins

    • MALDI MS: crystallize protein/peptide in matrix
    • Ionize w laser
    • Simpler analysis of mixtures of ions
    • Samples (proteins/peptides) are mixed with matrix and dried
    • Typically denatured samples
  80. Peptide fingerprinting
    • Defining protein identity by masses of its peptides
    • Many different peptides have nearly identical masses but the combination of masses is unique for each protein
    • Image Upload 16
  81. Peptide sequencing by tandem mass spectrometry (MS/MS)
    Image Upload 17
  82. MS/MS Sequencing
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  83. MS/MS analysis
    • Digest protein into peptides
    • MS1 observes different digested peptides- this alone can determine identity of the protein (peptide finger printing)
    • Select desired peptide (instrument control) break amide bonds per molecule in collision chamber
    • MS2: observe the series of peptide fragments
    • Use the known residue masses to determine sequence
Card Set
Biochem Ch 8 & 9