Protein Analysis|Graduate Biochemistry 6| Tulane

Purification and Analysis Overview

• Methods of protein separation and purification
• Protein solubility
• Isoelectric focusing
• Principles of chromatography
  • Polyacrylamide gel electrophoresis
  • Gel filtration
  • Ion exchange/Hydrophobic interaction
  • Affinity Chromatography
• Ultracentrifugation
  Methods of protein analysis
• Summary of primary information
• Protein sequencing by Edman degradation
• Mass spectrometry
• Methods of determining protein three dimensional structure

Methods of Macromolecule Separation

Method of Separation	Physical Basis	sensitivity	Specifity
Precipitation	Solubility (charge, isoelectric point)	μg - kg	Very low
Mass Spectrometry	Molecular Mass	fg - ug	Very High
Nondenaturing PAGE	Charge/mass, shape	μg - mg	High
SDS PAGE	Molecular Weight	μg - mg	High
Isoelectric focusing	Isoelectric point	μg - mg	Moderate
Capillary Electrophoresis	Charge/mass	ng - μg	High
Gel Filtration	Size and shape	ng - mg	High
Ion Exchange	Net charge	ng - mg	Moderate High
Hydrophobic interaction Chromatography	Hydrophobicity	ng - mg	High
Affinity Chromatography	Specific molecular interactoin	ng - mg	Very High
Density Gradient Ultracentrifugation	Molecular weight, density and shape	μg - g	Moderate
Equlibrium Density Ultracentrifugation	Density	μg - g	Moderate

Legend:

• Electrophoresis techniques
• Chromatography techniques
• Ultracentrifugation techniques

Protein Solubility in Salt

Salting in

• Increase in solubility with increasing salt at low ionic strength
• Proteins unfold at low ionic strength and irreversibly aggregate

Salting out

• Protein precipitation at high ionic strength driven by ionic charge shielding and solvent effects
• Solubility is minimum at isoelectric point (ionic effect)
• Effectiveness of salting out in Hoffmeister series (solvent effect):
  • Anions: SO4>H2PO4>CH3COO>Br>I>ClO4>SCN
  • Cations: NH4,Cs,K,Na>Li>Mg>Ca>Ba

PS: Increase the salt, you shield the charge surface of the protein

© Michel Awkal 2020	© Richard R Burgess 2009

Solting in: The unsaturated ions on the surface of the protein can interacted with other ions to increase the soluability of the protein.

Isoelectric focusing

© Blake C Meyers 2007

Isoelectric point (pI) is the pH at which positive and negative charges are equally abundant on a molecule.
pI for the G is ~6;
pI for D is ~3.5
pI for K is ~ 9.5

• Isoelectric focusing is equilibrium electrophoresis
• Ampholytes - Mixtures of buffer molecules covering a range of pKa values stabilize the pH gradient in an electric field
• Charged molecules, including the ampholytes, will move toward the location where their net charge is zero. This is the isoelectric point, or pI.

Chromatography

Principle of Chromatography

• Molecules in a mobile medium move through a stationary phase with which they can interact.
• Differential interactions with the stationary phase are the basis for physical separation of molecules

Some stationary phases

• Paper (paper Chromatography)
• Bare silica (Thin layer chromatography)
• Functionalized Silica (Column Chromatography)
• Various polymers (acrylamide, dextran, agarose, and others)

Mobile phase is usually liquid

SDS PAGE

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

Acrylamide gel:

• Polymerized acrylamide
• Macromolecule movement is hindered
• Movement driven by electric field
• Mobility is determined by charge, size and shape

SDS: A detergent that denatures proteins

• with/withough reducing agent:
  • breaking the disulfide bond

Proteins denatured in SDS:

• 3 SDS bound per amino acid
• Constant charge/mass
• Mobility α ln(mass)

• SDS can also solubilize hydrophobic AA
• Useful but may lots some information.

Gel Filtration Chromatography

© Marcell Wolf, 2015	© Rufika Shari Abidin

Vo = Void Volume, Excluded Volume
V_i = Included Volume
V_T (Total solvent volume) = V_o+V_i

For each mocromolecule
V_p = Penetrable volume
V_e (Protein Elution Volume) = V_o+V_p

σ (fractional retention) = V_p/V_i
V_p/V_i = (V_e - V_o)/(V_T - V_o)

• Polymer pours beets.
• separated the protein by its size
• You can’t tell the protein is monomor, dimer or etc.
• small proteins randomly filtered inside of the beets and causing transfer delay.

Ion Exchange Chromatography

• Retention is due to ionic interactions with fixed charges on stationary phase

• Counterions compete for binding sites

• Three phases: binding, elution, regeneration

Binding:

• Cation exchange: fixed anions
• Anion exchange: fixed cations

Typical Ionic groups

• Diaminoethylamino (DEAE)
• Carboxymethyl (caboxylate)
• Sulfoethyl/Sulfobenzyl (sulfate)

The material is charged:

• Same charge: not interacted
• opposite Charge: Reacted

Hydrophobic Interaction Chromatography

• Binding to stationary phase is hydrophobic
• Elution is with organic solvents (Acetonitrile, methanol, isopropanol, hexane)
• Often used in High Pressure Liquid Chromatography (HPLC).

Affinity Chromatography

• Retention is due to specific interaction

• Typical affinity molecules:

  • Antibody
  • Ligand/substrate/inhibitor
  • Lectin (carbohydrates)

• Nickel-Nitrilotriacetate or Ni-NTA Binds to engineered poly Histidine

PS: Some specific interact between the protein and the ?agent

Example of Enzyme Purification

Purification Step	Total Acitivity	% recovery	Specific Activity
Crude Extract	31800	100	1
Acid Precipitate	59900	189	10.1
Amm. Sulfate Precipitate	57200	175	27.2
GTP ( affinity)	37300	117	3962
Mono Q (ion exchange)	14300	45	6304
Gel filtration	11000	35	11620

Ultracentrifugation

Bouyant force depends on:

• Density Difference between molecule and solvent

Frictional force depends on:

• Molecular shape
• Solvent viscosity

Centrifugal force depends on:

• Molecular weight
• Rotation speed
• Distance from center

Ultracentrifugation

• Can be used to obtain information on molecular weight, molecular shape and intermolecular interactions
• Sedimentation velocity (S=Svedbergs)
• Sedimentation equilibrium

Density Gradient Ultracentrifugation

• Sucrose gradient:
  • Gradient is man-made and impermanent
  • Sample applied at top
  • Separation is by sedimentation velocity

CsCl density Gradient:

• Gradient is formed by centrifugal force
• Equilibrium position of macromolecule determined by its density

Protein Sequence Determination

Primary Information

• Number of chains
  • Dansyl Chloride reaction (Voet, Voet & Pratt pg 109)
  • One cycle of Edman degradation
  • SDS PAGE (under reducing conditions)
• Primary Sequence
  • Fragmentation, purification of fragments and Edman sequencing or mass spectroscopy
  • Requires overlapping fragments
    • Common fragmentation methods:
      • Cyanogen Bromide (cleaves C-term side of Met)
      • Trypsin (cleaves C-term side of Arg, Lys)
      • Chymotrypsin (cleaves C-term side of Trp, Phe, Tyr)
• Disulfide linkages
  • SDS PAGE (reduced and non-reduced)
  • Fragmentation, purification and partial sequencing with disulfide bonds oxidized/reduced

Protein Sequencing

Principles of Edman degradation:

• Cyclic, sequential removal and identification of N-terminal amino acids
• Identification is done by High Pressure Liquid Chromatography (HPLC)
• Can sequence 50-100 residues
• Sequence determination requires overlapping fragments

1. Derivitization: Phenylisothiocyanate (PITC)
2. Acid cleavage: PTC polypeptide terminal remove
3. Acid reduction: Polypeptide like of residue
4. Separation and Identification

(Back to 1.)
cite: Voet, Voet & Pratt pg 113

Mass Spectrometry

Principle of Mass Spectrometry

• Acceleration of a molecule in an electric field depends on the mass/charge ratio

Protein Mass Spectrometry
MALDI TOF - Matrix assisted laser desorption - Time of flight
ESI - Electrospray ionization

Both methods can be used to determine the molecular weight of proteins up to ~ 105 Daltons

Molecular weight determination is unambiguous

Proteomics - by mass spectrometry

Genomics: The study of the an organism’s genetic information.
Proteomics: The study of large scale protein expression pattern in a cell type or tissue.

Genomic information is fixed. Proteomic information depends on life cycle, environment, cell type etc.

Below: Two-dimensional gel electrophoresis of an E. coli whole cell extract.
Modern MALDI/SELDI mass spectrometers can identify these proteins
Often done by cutting out spots, digesting proteins with trypsin and getting the mass of the fragments by mass spec.

Determination of Protein Structure

• Two methods are used to determine protein structure:
  • X-ray crystallography
  • Two-dimensional Nuclear Magnetic Resonance (NMR) spectroscopy

X-ray crystallography

• purify protein
• grow crystals
• collect X-ray diffraction patterns
• determine electron density in three dimensions
• solve protein structure

Advantages:

• Can solve very large proteins/complexes
• Gives atomic-level resolution

Disadvantages:

• crystallization is often difficult or impossible
• crystal contacts can distort structure

Determination of Protein Structure

2D NMR Spectroscopy

• purify protein
• collect multidimensional NMR spectra in solution
• assign peaks and crosspeaks to specific residues
• calculate distances between residues from the strength of the crosspeaks
• solve structure using distance constraints
  Advantages:
• no crystallization required
• structure is a solution structure
  Disadvantages:
• Only small proteins (<200 residues) can be solved
• Resolution is not at atomic level