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• Protein purification involves breaking open cells, homogenizing the cell lysate, and pelleting organelles and other large objects through centrifugation.
• Particles reaching the tube's bottom via centrifugation form a pellet.
• Smaller particles remain in equilibrium in the supernatant.
• Larger objects drift faster during centrifugation.

Ion-Exchange Chromatography

• Separates proteins based on the sign and magnitude of their net electric charge.
• Clear lysate is passed through a bead-packed column.
• Resin used is either cation- or anion-bound charged groups.
• A low-salt buffer washes the column to remove unbound proteins.
• Increasing free salt ions reduces electrostatic interaction between the resin and bound protein.
• The salt concentration is gradually increased as elutents are collected.

Size-Exclusion (Gel Filtration) Chromatography

• This separates proteins based exclusively on their size.
• The resin contains porous beads with labyrinth-like paths.
• Molecules near the pore size get trapped and migrate slowly.
• Large molecules pass freely.
• The lysate is loaded without washing.
• The column is eluted by flowing a buffer solution.
• Large proteins emerge from the column before small proteins.
• Size-exclusion chromatography, also known as gel filtration, separates proteins based on size; larger proteins emerge sooner.

Affinity Chromatography

• Separates proteins based on binding affinity.
• The protein of interest binds to the column.
• Other proteins are washed away.
• The desired protein is eluted using a high concentration of salt or a ligand that competes with the protein's binding.
• Affinity chromatography, the use of calcium attached to beads, allows for more rapid flow through of proteins without calcium-binding properties.

Protein Purity & Specific Activity

• Purification steps increase the purity of the protein.
• Purification steps also increase the specific activity of the protein. The purification factor is the final specific activity divided by the starting specific activity.

Gel Electrophoresis

• Used to measure molecular weight, purity, and oligomeric state of proteins and nucleic acids.
• The sample is loaded into wells of a polyacrylamide gel (typically a 2-D gel).
• An electric field is applied to force migration into the gel.
• Proteins separate by their electrophoretic mobility, dependent on mass, net charge, and shape.

Electrophoretic Mobility

• Described by the equation μ = Vdrift/E = Ze/f, where Z is the total net charge, E is the electric field.
• For the equation, f is the friction coefficient, and Vdrift is the drift velocity.
• Native gels do not denature the proteins.
• The direction and speed of mobility depend on both the size (f) and net charge (Z).
• Issues arise because proteins vary greatly in their charge-to-mass ratio
• Proteins with a zero net charge do not migrate into the gel, while proteins with a net positive charge migrate in the opposite direction.

Denatured Gels

• Proteins are denatured by heat and the detergent SDS.
• SDS binds to the protein at a rate of approximately one SDS molecule per two amino acids creating a net negative charge proportional to the length (i.e. mass) of the peptide chain.
• The mass-to-charge (m/z) ratio becomes roughly the same for all proteins.
• Polyacrylamide reduces the mobility of an unfolded chain linearly by the log Mr of the proteins.
• Oligomers or complexes split into individual peptide chains during electrophoresis.

Blue Native Gels

• Nonspecifically coat proteins with a negatively charged molecule (Coomassie G-250) without denaturation.
• Converts protein molecules to a net negative charge through the gel.
• Complexes are not disassembled; their mass, abundance, and purity can be determined.

Absorption Spectroscopy: Beer-Lambert Law

• The intensity of transmitted light through a sample can be used to determine its absorption properties using the Beer-Lambert Law
• Transmission T = Io / I0, where T decreases exponentially by path length as T=e^(-εcl)
• Absorbance A= -logT = -log(e^(-εcl)) = εcl
• The specific absorbance of a protein or nucleic acid is measured relative to a blank buffer.

Measuring Protein Concentration from Absorbance

• Major contributions to protein absorption at 280 nm stem from aromatic tryptophan (W) and tyrosine (Y) residues which have high extinction coefficients
• Phenylalanine (F) has lower maximal absorbance at 260 nm
• The extinction coefficient at 280 nm (ε280) can be calculated using the formula: ε280 = nw x 5,500 + ny x 1,490

Mass Spectrometry

• Used to analyze molecules by ionizing the sample.
• Electrospray ionization involves passing molecules through a needle to disperse the solution into a fine mist of charged droplets.
• The needle is kept at a high voltage, causing positively charged ions (H+ or Na+) from the solution to be added to droplets.
• The solvent rapidly evaporates in a vacuum, leaving multiply charged macromolecular ions in the gas phase.

Time-of-Flight (TOF) Mass Spectrometry

• This separates ions by their mass-to-charge ratio (m/z).
• Ions are accelerated linearly through an electric field in the gas phase.
• Ions with a lower m/z will be accelerated more.
• m/z is deduced from the time of flight of the ion before it hits the detector.

Mass Spectrometry: Orbitrap

• Ions are deflected vertically by a magnetic field (dependent on their masses).
• Ions with lower m/z deflect more from their path.
• The electron's trajectory (where the ion hits the detector) is converted to m/z.

Mass Spectrum of a Whole Protein

• A whole protein ionized using electrospray ionization produces peaks representing different charges but the same mass
• Adjacent peaks differ by a charge of +1.
• This determines the mass of the protein.

Mass Spectrum of a Cleaved Protein

• This is often done via proteolytic digestion (most commonly with trypsin)
• Protein is cleaved into fragments to be ionized using MALDI
• The positive ions predominately carry a charge of z = +1.
• All major peaks have the same charge but differ by mass.
• This measures molecular mass with 1 Da accuracy.

Peptide Sequencing: Tandem MS

• MS-1 sorts peptides produced by proteolytic cleavage, with only one peptide emerging.
• The collision chamber fragments the peptide further into two pieces (b type and y type) by breaking a peptide bond.
• MS-2 measures m/z ratios of charged fragments.
• Each peak measures m/z ratios of charged fragments from the second MS.
• Mass differences between successive peaks reveal the peptide sequence.
• Mass spectrometry can be used to monitor changes in the cellular proteome as a function of metabolic state.

Determining Protein Structure: Cryo-Electron Microscopy

• The sample is flash-frozen in vitreous (noncrystalline) thin ice.
• A high-intensity electron beam is focused onto the flash-frozen sample.
• The transmitted beam is focused onto a direct electron-detector camera.
• 2D images of ~100,000 individual molecules are reconstructed to obtain the 3D structure.

Bioinformatics: Sequence Alignment

• Amino acid sequence can inform 3D structure, function, cellular location, and evolution.
• Proteins with similar functions (homologs) have regions with well-conserved sequences.
• Homologs in the same species are called paralogs; homologs in different species are called orthologs.
• Sequence alignment tools assign a score based on two proteins with identical segments (positive score) or gaps (negative score).
• The consensus sequence reflects the most common amino acids at each position among homologous proteins.
• Overall height in sequence alignment reflects sequence conservation.
• The height of individual symbols reflects the frequency of that amino acid.
• Amino acids with similar characteristics (charge, polar, nonpolar) are often interchangeable within a sequence.