MBIO 2700 / Topic 5b: Proteins in the Lab
Studied by 30 people
Learn
Practice Test
Spaced Repetition
Match
Flashcards1/15
There's no tags or description
Looks like no tags are added yet.
Name | Mastery | Learn | Test | Matching | Spaced |
|---|
No study sessions yet.
16 Terms
What are the three steps to the Sanger protein sequencing? Define each step.
(1) Fragmentation: Polypeptide hydrolysis into small fragments.
(2) Sequencing: Separating small fragments + determining AA seq of each.
(3) Alignment: Alignment of small sequences to generate once-complete sequence.
Why can’t we directly determine the amino acid sequence of the polypeptide as a whole instead of hydrolyzing and separating it into polypeptide fragments first?
> Polypeptides aren’t optimal for sequencing as a whole.
> Smaller fragments = better → Chop into smaller fragments of polypeptide.
What’s the downside of chopping the polypeptide up?
Losing the AA seq and have to re-align it to get lost sequence.
What are the three enzymes usually used in fragmentation, and what does each cleave?
• Trypsin: Cleaves after Lys or Arg (basic residues).
• Chymotrypsin: Cleaves after Phe, Tyr, or Trp (aromatic residues).
• Cyanogen bromide (CNBr): Cleaves after Met residues.
How are enzymes used in fragmentation usually perform the whole cleaving process?
> E binds to S (the AA/residue).
> E cleaves off bond at carboxyl side of residue.
How is sequencing usually performed?
> Sanger protein sequencing uses a process called Edman degradation.
> Edman degradation uses phenyl isothiocyanate = derivatized AA that can be identified by chromatography.
What does a mass spectrometer measure? How does it measure what it measures?
Mass of atoms and molecules by ionizing them from a sample.
Why do we need to ionize particles?
> Manipulate particles via electric and magnetic fields of spectrometer.
> Separate particles based on their mass-to-charge ratio.
Why does the charge on all ions have to be the same in mass spectrometry? What happens if we have different charges on ions?
> If charge on all ions = same → mass-to-charge ratios comparable btwn diff ions.
> If charge on all ions = different → different q’s = different reference points → mass-to-charge ratios incomparable btwn ions.
• What is “time of flight” in mass spectrometry?
• What’s the relationship between mass and time of flight?
• Time of flight = measurement of time for ions to move through field + reach detector.
• Relationship = Mass ↑ Time of flight ↑
• What is protein purification?
• What are the four ways you can purify proteins by?
• Protein purification = Isolation of a specific protein from a complex mixture.
• Four ways are:
> By charge. IEX chromatography.
> By size. SEC chromatography.
> By solubility.
> By affinity for materials. Very specific. Affinity chromatography.
Can you explain how ion exchange chromatography (IEX chromatography) works for a cation exchanger?
> pHsol < pIpeptide of peptide in column.
> Peptides are (+).
> (+) peptides will attach to resin beads that are (-)-charged.
> All other unwanted peptides elute.
ROUTE ONE:
> pHsol ≥ pIpeptide of peptide in column.
> Peptides are neutral/negative.
> Peptides detach from resin beads.
ROUTE TWO:
> Add salt.
> The (-) ions of the salt will elute down, but the (+) charged ions will compete with the peptides, i.e. (+)-charged peptides vs. (+)-charged ions for the resin bead spot.
> (+) charged ions win the bead spots, and peptides go home (elute down the column and are collected).
Can you explain how ion exchange chromatography (IEX chromatography) works for an anion exchanger?
> pHsol > pIpeptide of peptide in column.
> Peptides are (-).
> (-) peptides will attach to resin beads that are (+)- charged.
> All other unwanted peptides elute.
ROUTE ONE:
> pHsol ≤ pIpeptide of peptide in column.
> Peptides are neutral/positive.
> Peptides detach from resin beads.
ROUTE TWO:
> Add salt.
> The (+) ions of the salt will elute down, but the (-) charged ions will compete with the peptides, i.e. (-)-charged peptides vs. (-)-charged ions for the resin bead spot.
> (-) charged ions win the bead spots, and peptides go home (elute down the column and are collected).
How does size-exclusion chromatography (SEC chromatography) work?
Also known as gel-sieving chromatography.
> Porous beads are packed in the column.
> Small peptides must pass through the beads, getting into heavy “traffic,” because they have no choice. This heavy traffic causes small peptides to go elute slowly. It is very unlikely for small peptides to pass around the traffic.
> Big peptides do not fit in the pores of the beads, and pass around the traffic, causing them to elute faster.
How does affinity chromatography work?
> Attach ligands to beads in column.
> Mixture of peptides are added to the column.
> Ligands on the beads attach to the binding site of the peptides the ligands are specific to.
> The rest of the peptides are eluted out of the column.
> The competitors are added to the column, fighting the ligands for the binding sites of the protein, i.e. ligands vs. competitors.
> Competitors win and take the proteins as their trophies and go home (they exit the column).
> What does sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) measure?
> How does SDS-PAGE work?
• SDS-PAGE = estimates mass of protein.
• It works in this series of steps:
> SDS binds to proteins → gives them uniform negative charge + denaturing them.
> Polyacrylamide gel = molecular sieve → smaller proteins move faster.
> Electric current applied → (-) charged proteins migrate towards (+) electrode.
> All proteins have uniform charge → separate based on size, w/ smaller proteins moving faster.
> Bands visualized by staining with Coomassie Blue or Silver.