Lecture 12 (Protein Purification, Detection & Mass Spectrometry)

What are the 3 major components of protein purification?

1. Solubilization

2. Separation

3. Protein detection

Why is E. coli often used for protein expression?

Engineered E. coli can overexpress high yields of a protein of interest

What is a limitation of protein overexpression?

Engineered cells may lack required post-translational modifications for function

What is cell lysis?

Breaking open cells to release proteins, forming a crude extract/cell lysate

What are common cell lysis methods?

• Osmotic shock = Cells in hypotonic solution take up water and burst

• Mechanical:

  • Sonication = Sonication uses ultrasound

  • French press = Push with high pressure through a small hole

What factors stabilize purified proteins?

• Correct pH (can easily denature by changes in pH → need buffers)

• Cold temperature

• Protease inhibitors (proteases can destroy protein of interest)

• Detergents (hydrophobic proteins need to be kept soluble)

What is the pellet vs supernatant in centrifugation?

• Pellet = insoluble material

• Supernatant = soluble fraction (where protein of interest resides)

What is differential centrifugation?

Sequential spins at increasing speed to pellet different organelles/fractions

What is isopycnic centrifugation?

Organelles sediment in a density gradient until buoyant density matches

What is protein precipitation used for?

• Reduce solubility

• Pellet

• Concentrate the protein

• Re-dissolve proteins

What is salting in vs salting out?

• Salting in keeps proteins soluble

• Salting out uses high salt to precipitate/concentrate proteins

What is an ammonium sulfate cut?

Protein precipitation/concentration using ammonium sulfate

What is dialysis used for?

Semipermeable membrane removes small molecules like salt while retaining proteins

What is chromatography?

Separation/fractionation based on physicochemical properties

What are mobile and stationary phases?

• Mobile phase = protein solution (contains protein of interest in solution)

• Stationary phase = column matrix (moving solution through column of packed porous solid material)

What is retention time?

Time/fraction when a molecule elutes from a column

What is ion exchange chromatography?

Separates proteins by charge using charged stationary-phase resin

What do anion vs. cation exchangers bind?

• Anions exchangers:

  • Anions

  • Resin is positively charged

• Cation exchangers:

  • Cations

  • Resin is negatively charged

How does protein charge affect ion exchange choice?

Proteins are polyelectrolytes (have both negative/positive charges and their net charge depend on pH)

• Acidic proteins → anion exchange

• Basic proteins → cation exchange

How do proteins elute in ion exchange?

• Lower-affinity proteins elute faster

• High salt competes off stronger binders

What is gel filtration chromatography?

Size-exclusion chromatography that separates proteins by size

• Dilutes the sample

How do large vs small proteins behave in gel filtration?

• Large: Cannot enter beads → small void volume → elute first

• Small: Enter bead pores → larger volume → elute later

What is affinity chromatography?

Stationary phase binds only the target protein, allowing one-step purification

What is the most common affinity tag?

Six-His residue tag at the N- or C-terminus

How does nickel affinity chromatography work?

• His-tag binds Ni2+ resin (stationary phase)

• Imidazole competes off and elutes the protein

How are chromatography fractions collected?

As fractions using a fraction collector

What absorbs at 280 nm?

Mostly Trp and Tyr, plus a little Phe

• W > Y > F

What absorbs at 214 nm?

Peptide bonds

What is electrophoresis?

Separation of charged proteins by migration in an electric field

What is PAGE?

• Polyacrylamide gel electrophoresis

• Gel acts as a molecular sieve

  • Slows migration of proteins based on their charge-to-mass ratio and their shape

What can electrophoresis reveal?

• Protein purity

• Approximate molecular weight

• Isoelectric point (pH at which net electric charge = 0)

What is SDS-PAGE used for?

• SDS = Sosium dodecyl sulfate

• Estimating purity and molecular weight

• Uses heat to denature proteins and gives large negative charge

How can SDS-PAGE bands be identified?

Excise band → digest into peptides → identify by mass spectrometry

What do proteases and chemicals do in protein ID?

Cleave internal peptide bonds next to specific amino acids

What is mass spectrometry?

Measures mass-to-charge ratio (m/z) of gas-phase ions

What is MS/MS used for?

Peptide sequencing/identity confirmation by tandem mass spectrometry

What are the 2 main ionization methods?

• MALDI

  • Protein + matrix is laser-ejected into gas phase, usually +1 charge

  • Time-of-flight (TOF) depends on mass and charge (m/z)

• ESI

  • High-voltage spray makes charged droplets

  • Evaporation yields gas-phase protein ions

Why is ESI often more accurate than MALDI?

Multiple charge states allow a mass average from many m/z species