Lecture 11 - Protein Sequencing

Question 1

Composition of a Protein

Answer 1

• Gives all the amino acids in the protein
• No sequence information
• Hydrolyze all amide bonds and separate amino acids by chromatography

Question 2

End-Group Analysis

Answer 2

• Allows us to determine the identity of the amino acid at the N-terminal or C-terminal of a peptide/protein
• use dansyl chloride for N-terminal (also reacts with lysine side chains)
• Use enzymes called carboxypeptidases for C-terminal

Question 3

Exopeptidases

Answer 3

Enzymes that cleave off amino acids at either terminus

Question 4

Endopeptidases

Answer 4

Enzymes that cleave off amino acids somewhere in between the termini

Question 5

Disulfide Bond Cleavage

Answer 5

• Cystine residues can interfere with sequencing procedures
• Use b-mercaptoethanol to reduce cystine to cystine interaction

Question 6

Edman Sequencing

Answer 6

• Generates amino acid sequence from the N-terminal end
• Can only sequence 30-50 amino acid residues
• Need a method to break larger proteins into smaller peptides
• ## Cannot sequence through disulfide bonds

Question 7

Trypsin

Answer 7

• Enzyme used as reagent to make smaller peptide fragments
• Hydrolyze to the right of specific residue
• Cleaves after positively charged amino acids as long as long as they aren’t attached to Proline

Question 8

Chymotrypsin

Answer 8

• Enzyme used as reagent to make smaller peptide fragments
• Hydrolyze to the right of specific residue
• Cleaves after aromatic residues as long as there is no Proline attached to the aromatic residue

Question 9

Protein Sequencing Steps

Answer 9

• Purify Protein
• Treat with B-mercaptoethanol
• Treat separately with trypsin, chymotrypsin, or CNBr
• Separate the peptides by chromatography
• Sequence each peptide by the Edman sequencing method
• Analyze the data to generate the intact peptide

Question 10

Mass Spectrometry

Answer 10

• Short peptide sequences can also be sequenced by mass spectrometry

Question 11

Bioinformatics

Answer 11

Interdisciplinary field that develops methods and software tools for understanding biological data