PHAR505 LEC 8

Why purify proteins?

• Obtain enough pure protein for structure determination: mixed samples hinder crystallization, and impurities interfere with NMR signals.

• Measure drug–protein binding accurately using methods such as SPR or fluorescence anisotropy → reliable Kd

• Measure specific activity = enzyme activity per unit mass of protein.

• Remove other enzymes whose activity could be mistaken for the target protein’s activity.

How does liquid chromatography separate proteins?

• Mobile phase: Buffer pumped continuously through a column

• Stationary phase: Tightly packed, porous resin beads with selected chemical properties; pores provide a large surface area.

• Proteins interact differently with the beads → leave the column (elute) at different times in the buffer.

• Track elution using fluorescence, conductivity, or absorbance against time or buffer volume.

• Steps: Equilibrate column → load sample → wash away unbound material → elute the desired protein.

How does size-exclusion chromatography (SEC) separate molecules?

• Uses porous,agarose beads; buffer carries molecules through the column.

• Large molecules: Cannot enter pores → travel between beads (void volume) → elute first.

• Medium molecules: Enter larger pores → delayed → elute next.

• Small molecules: Enter both small and large pores → most delayed → elute last.

• Elution depends on the space accessible to each molecule.

• Uses one unchanged buffer throughout

What can SEC tell us, and what conditions improve separation?

• Elution volume relates linearly to log (molecular weight) within the beads’ separation range.

• Preserves proteins’ native structure, helping identify subunit assembly: a dimer has twice a monomer’s mass.

• Shape also affects elution: Compact, globular proteins enter pores more easily → elute later than similarly sized extended proteins.

• Use a small sample volume and a long column to keep peaks narrow.

• Choose beads suited to the desired molecular-weight range.

• Allows buffer exchange and desalting: small buffer components and salts elute later than proteins.

How can SEC detect protein oligomers, such as antibody hexamers?

• Proteins can join through noncovalent interactions to form larger assemblies.

• Larger assemblies elute earlier in SEC.

• Increased antibody hexamer formation produces a larger early-eluting peak; these assemblies can trigger an immune response.

How does reverse-phase chromatography separate compounds?

• Mainly purifies small organic molecules and peptides (<10 kDa).

• Stationary phase: Nonpolar silica beads, often coated with C18 carbon chains.

• Mobile phase: Water with acid, such as 0.1% formic acid; denaturing conditions expose hydrophobic regions.

• Less hydrophobic compounds bind weakly → elute earlier; more hydrophobic compounds bind strongly → elute later.

• Increase a water-miscible organic solvent, such as acetonitrile or methanol, to release bound compounds.

• Volatile solvents allow coupling to electrospray ionization (ESI) mass spectrometry

• In reverse-phase chromatography: The acid keeps the sample at a low, consistent pH, helping control the molecules’ charge and produce clear peaks. The acidic conditions plus organic solvent can also unfold proteins, exposing hydrophobic parts that interact with the beads.

  In SEC: We usually want proteins to stay folded, so we use a gentle, water-based buffer containing:

Why are molecules ionized in mass spectrometry, and what methods are used?

• Charge allows electric/magnetic fields to separate ions and determine their mass-to-charge ratio (m/z).

• EI (electron ionization) and FAB (fast atom bombardment): Mainly small molecules.

• MALDI (matrix-assisted laser desorption/ionization) and ESI (electrospray ionization): Commonly proteins.

How does electrospray ionization (ESI) work, and what are its advantages and limitations?

• Liquid leaving an LC column passes through a nozzle with nebulizer gas, forming tiny droplets.

• High voltage charges the droplets; hot drying gas evaporates the solvent.

• Charge repulsion breaks droplets apart, releasing individual gas-phase ions, often positively charged and carrying multiple charges.

• Electric fields guide ions into the mass analyzer.

• Advantages: Sensitive, little fragmentation, handles large molecules >100 kDa.

• Limitations: Sensitive to salt; multiple charge states complicate the spectrum.

Why does one protein produce multiple peaks in a mass spectrum?

• Peaks represent different charge states of the same protein.

• Charge state = number of bound protons.

• Each charge state has a different m/z.

• Used to determine the protein’s molecular weight.

How does resolution affect isotope peaks?

• Higher resolution: separates individual isotope peaks.

• Monoisotopic mass: calculated using one specific isotope of each element, usually the lightest.

• Adjacent isotope peaks differ by approximately 1 Da in mass (1/z in m/z).

• Isotope patterns can indicate compound origin, e.g., doping tests.

How does tandem MS (MS/MS) work?

• MS1: measures parent ions and selects one.

• Collision cell: selected ion collides with inert gas, such as nitrogen.

• Collision-induced dissociation (CID): breaks labile bonds, e.g., peptide bonds.

• MS2: measures fragment m/z to help determine molecular structure.

How is LC–MS/MS used to identify and quantify drugs using multiple reaction monitoring (MRM)?

• Detects drugs and metabolites in complex mixtures by monitoring parent ion → fragment ion transitions.

• First run standards of known concentration through C18 LC–MS/MS to determine each drug’s fragmentation pattern.

• Characteristic mass transitions confirm the drug’s identity, helping distinguish molecules with the same m/z.

• Compare sample ion intensities with standards to determine drug concentrations.

• MRM monitors transitions from multiple parent ions.

It rapidly switches between monitoring these two parent → fragment pairs, measuring how much of each molecule is present. That’s why it’s called multiple reaction monitoring (MRM).

How does LC–MS/MS quantify imatinib and its metabolite, N-desmethyl-imatinib?

• The liver metabolizes imatinib into N-desmethyl-imatinib.

• MRM monitors both molecules’ parent → fragment transitions in parallel to quantify their plasma levels.

• Each parent ion is isolated, then fragmented in a collision cell by breaking its most labile bond—the bond that breaks most easily.

• Unfragmented parent ions appear at m/z 494 for imatinib and 480 for its metabolite.

• Both produce a major daughter/fragment ion at m/z 394, but their different parent ions distinguish them: 494 → 394 versus 480 → 394.

• Reverse-phase LC separates the molecules; MRM chromatograms show each transition’s signal intensity over elution time.

• Compare chromatogram peak areas with known standards to determine concentrations.

• Plot plasma concentration against time after dosing to study their pharmacokinetics.

How does LC–MS/MS identify proteins in a sample?

• Digest proteins into peptides using an enzyme such as trypsin

• Separate peptides on a C18 reverse-phase column

• Eluted peptides undergo electrospray ionization (ESI) and enter the mass spectrometer.

• MS1: records parent-ion m/z and isotope patterns at each point during elution.

• Select parent-ion peaks and fragment them.

• MS2: measures the resulting daughter ions; their fragmentation patterns help identify peptide sequences, with identification scores.

• Use the identified peptides to determine which proteins are present, allowing identification of multiple proteins in one sample.

Differences between the 2 exps

The imatinib example tells us how much drug is present; the protein example tells us which proteins are present.

• Imatinib: We already know the molecules we’re looking for. Monitor their known parent → fragment transitions and use peak areas to calculate concentrations.

• Protein identification: We don’t know which proteins are in the sample. Analyze peptide fragmentation patterns to identify their amino acid sequences, then match those peptides to proteins.

Both use LC–MS/MS, but they answer different questions: “How much?” versus “Which proteins?”