Protein Separation & Purification Methods

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Protein Separation and Purification Methods

• Solubility uses salting out.
• Ionic Charge uses ion exchange chromatography, electrophoresis, and isoelectric focusing.
• Polarity uses hydrophobic interaction chromatography.
• Size uses gel filtration chromatography and SDS-PAGE.
• Binding Specificity uses affinity chromatography.

Proteomics - 2D Gel Electrophoresis

• This analysis compares two sets of cells, one healthy and one cancerous.
• Orange proteins are present in healthy cells but not in cancerous ones.
• Blue proteins are found in cancerous cells, but not healthy cells.
• Black proteins are equally abundant in both healthy and cancerous cells.

Ultracentrifugation

• Separates macromolecules by mass.
• Ultracentrifuges can spin at speeds of up to 150,000 revolutions per minute (RPM) and 1,000,000 x g.

Protein Purification Process

• This a multi-step approach, to purify and enrich the protein of interest.
• Quantifying protein can be achieved by measuring its unique character.
• To quantify an enzyme, measure its activity through an assay.

Protein Purification Table breakdown for a Hypothetical Enzyme

• Step 1: Crude cellular extract; volume: 1,400 ml; total protein: 10,000 mg; activity: 100,000 units; specific activity: 10 units/mg.
• Step 2: Precipitation with ammonium sulfate; volume: 280 ml; total protein: 3,000 mg; activity: 96,000 units; specific activity: 32 units/mg.
• Step 3: Ion-exchange chromatography; volume: 90 ml; total protein: 400 mg; activity: 80,000 units; specific activity: 200 units/mg.
• Step 4: Size-exclusion chromatography; volume: 80 ml; total protein: 100 mg; activity: 60,000 units; specific activity: 600 units/mg.
• Step 5: Affinity chromatography; volume: 6 ml; total protein: 3 mg; activity: 45,000 units; specific activity: 15,000 units/mg.
• After step 5, the enzyme has been purified by a factor of 1,500.
• The yield of the enzyme is 45%.

Definitions for Protein Purification

• Total protein refers to the quantity of total protein in a fraction, measured in mg or g.
• Total activity refers to the quantity of the target protein measured by its activity/function.
• Need to use an assay for this; total activity is measured in the fraction.
• Specific Activity is total activity divided by the amount of protein, measured in units of activity/mg of protein.
• Yield measures the total activity retained after each purification step as a percentage of the total activity in the initial homogenate.
• Purification level (or fold purification) measures the increase in purity which can be obtained by dividing the specific activity by the specific activity of the initial homogenate.

Example using Pepsinogen C Data

• Data in units of (mg), (units), and (units/mg).
• Crude extract: 130, 11,500, 88.5
• Salting out: 23, 7,980, 347
• Ion exchange: 11.4, 5,130, 450
• Gel filtration: 1.6, 750, 457
• Yield is the total activity retained after each purification step/total activity in the initial homogenate X 100.
• Fold purification refers to specific activity at purification stage/specific activity of the initial homogenate.
• Specific activity is determined by activity divided by total protein and increases as purity increases.
• Yield percent for Pepsinogen C at the gel filtration step is 6.5%.
• Fold purification at gel filtration step is 5.16-fold.

Protein Sequencing

• It determines the amino acid sequence of a protein.

Determining the Primary Structure of Proteins

• Start with a purified protein sample.
• Convert into a single chain if disulfide bonds are present.
• Cut into short fragments using more than 2 enzymes (endo peptidases) or chemicals.
• Run chemical reactions to figure out the order of amino acids in the fragments.
• Can use Dansyl Chloride (N-terminal amino acid only), or Edman degradation (sequencing from N-terminal).
• Assemble the fragments into correct order

Cutting Di-Sulfide Bonds

• Cutting di-sulfide bonds is a concept used in identifying proteins.

Amino-Terminal Analysis with Dansyl Chloride

• This reacts with a primary amine.
• Understand the basic concept, no need to memorize structures for dansyl chloride or dansyl-amino acids.

Edman Degradation

• Cleaves off the first amino acid on the N-terminal and derivatizes it to deduce the sequence from N-terminal to C-terminal by the order of appearance of the derivatized amino acid residue.
• Leaves the rest of the peptide unhydrolyzed for more rounds of reaction.

Peptides sequenced by mass spectrometry

• It makes use of HPLC Analysis
• It involves Electrospray Ionization (ESI) MS

Endopeptidases

• Important in protein characterization.

Specificity of various endopeptidases

• Trypsin cleaves when R(n-1) is a positively charged residue (Arg, Lys), and R(n) is not Proline.
• Chymotrypsin cleaves when R(n-1) is a bulky hydrophobic residue (Phe, Trp, Tyr); R(n) not Proline. Cleaves more slowly for Rn–1 = Asn, His, Met, Leu
• Elastase cleaves when R(n-1) is a small neutral residue (Ala, Gly, Ser, Val); R(n) not Proline.

Amino acid sequencing using protease

• Sample peptide: 11 amino acid residues (undecapeptide).
• When digested with Trypsin one gets Ser-Phe-Gly-Arg, Val-Leu-Lys, and Tyr-Ala-Gln-Thr.
• When digested with Chymotrypsin one gets Ala-Gln-Thr, Gly-Arg-Tyr and Val-Leu-Lys-Ser-Phe.
• Amino acid before the scissile bond for Chymotrypsin is Phe, Trp, Tyr, for Trypsin it's Arg, Lys, and for Cyanogen bromide (chemical cleavage) its Met.

Cyanogen Bromide

• Cyanogen bromide cleaves at Met residues

Protein Sequences and Relationships

• Protein sequences reveal evolutionary relationships.

Example Question to Determine Amino Acid Sequence

• Kara isolated a peptide and runs one cycle of Edman degradation.
• Treatment with cyanogen bromide yields four fragments: Lys-Pro-Ala-Met, Pro-Asp, Leu-Ile-Cys-Trp-Met, Thr-Arg-Met.
• Treatment with chymotrypsin yields Met-Pro-Asp.
• Determine the amino acid sequence using the various peptide treatments, placing the fragments in order.

Protein Structures (Overview)

• Primary structure is the amino acid sequence.
• Secondary structure involves α helices and β sheets whose driving force facilitates the secondary structure.
• Tertiary structure involves 3D folding whose driving force dictates the relationship between the tertiary structure vs. secondary sequence (of amino acids).
• Quaternary structure has subunits (e.g., hemoglobin/RNA polymerase).

Atoms in Peptide Bonds

• Electronegativity measures a tendency to attract electrons.
• The values are H (2.1), C (2.5), N (3.0), O (3.5).
• Differences in electronegativity change electron distribution.

Peptide Bonds

• Resonance structure stabilizes the peptide bond
• Resonance causes a “double bond”.
• There is C=O or C=N in the peptide bond.
• Makes it rigid (40% double bond).
• It has planar configuration among the atoms in the peptide bond.

Trans vs Cis Conformation

• In trans configuration, the two alpha carbon atoms of the connected amino acids are on the opposite sides of the peptide bond.
• In cis configuration, the alpha carbons are on the same side of the peptide bond.
• Most peptide bonds in proteins are trans.
• Trans is favored because there is potential steric hindrance with the cis conformation.

Dihedral Angles

• Main chain conformations are use to determine the secondary structure
• Dihedral angles are located on the two planes
• Ф (Phi) Is located in between N and Cα which form the (hetero-atoms)
• Y(Psi) Which Is located in between Cα and C (same-atoms)
• The third angle is (ω) located around N and C form the (peptide bond)

Ramachandran plots and allowed Conformations

• It reveals the distribution of ɸ and Ψ dihedral angles that are found on a protein.
• It shows the common secondary structure elements
• It reveals regions with unusual backbone structure
• Blue-shaded regions = sterically allowed ɸ and Ψ angles for all residues except Gly and Pro.
• Green-shaded regions indicate the more crowded (outer limit) ɸ and Ψ angles.
• Yellow circles are conformational angles of several secondary structures: -right-handed α helix, parallel β sheet, antiparallel β sheet, C, collagen helix, and a, left-handed α helix.
• Most areas represent forbidden conformations of a polypeptide chain and there are 3 small regions that are physically accessible to most residue.
• There are however some exceptions, most values accurately determined by structure.