determining amino acid sequence of a protein

Question 1

fluorodinitrobenzene

Answer 1

bright yellow tag that identifies first amino acid in a protein (N-terminus)

Question 2

sanger method

Answer 2

method for determining the first amino acid in a peptide chain, destroys the rest of the chain
- found at AA N-terminus

Question 3

Edman degradation

Answer 3

AA sequencing method that allows N-terminal amino acid to be reacted, removed and identified without hydrolyzing the peptide bonds
- can sequence up to first 50 AAs

Question 4

steps to cleave peptide bonds in Edman degradation

Answer 4

1. Coupling (basic phase)
2. Cyclization (acidic phase)

Question 5

Coupling

Answer 5

requires a base, labels the N-terminal AA with PITC
- once completed cyclization can take place

Question 6

Cyclization

Answer 6

requires acid, sulfur or PITC does nucleophilic substitution and reacts with carboxyl group on first AA to cleave peptide bond
- once completed next coupling can take place

Question 7

Amino acid sequencing through molecular biology

Answer 7

DNA sequencing: can predict the protein sequence using the genetic code

Question 8

selective hydrolysis

Answer 8

cuts polypeptide at specific locations to yield ogliopeptides

Question 9

trypsin

Answer 9

digestive enzyme that binds Arg or Lys side chains by their carboxylate group - proline has the wrong shape and won’t fit in position after Arg or Lys

Question 10

At which end do fragments have the amino acids where the bonds were cleaves

Answer 10

C-terminal end, except the fragment at the C-terminus

Question 11

Chymotrypsin

Answer 11

enzyme that binds and cleaves peptide bonds Phe, Try or Trp
- except when proline follows

Question 12

Cyanogen bromide

Answer 12

attacks the S atom of Met, chain is broken on carboxylate side and Met is converted to Hse, will still cleave bond if followed by Pro since it is a chemical reaction

Question 13

overlap method

Answer 13

• two samples of original peptide are cut by 2 different hydrolysis methods targeting different sites
• sequences from one set of oligopeptides are lined up to overlap with oligopeptides from another set to deduce how they were originally joined

Question 14

What amino acid is found at the C-terminus when peptide is cut by trypsin

Answer 14

K or R

Question 15

Peptides generated at low pH

Answer 15

• acidic residues have no charge on side chain (COOH)
• basic residues have a +1 charge on side chain (NH3+)
  peptides with charges produce the highest signals

Question 16

MS fragmentation of peptide

Answer 16

reassemble peptides starting from the C-terminus, then flip so it is in core N to C terminus order