Peptide sequencing is crucial for understanding protein structure and function. Techniques like Edman degradation and partial hydrolysis allow scientists to determine the order of amino acids in peptides and proteins, providing valuable insights into their biological roles.

Enzyme specificity plays a key role in peptide cleavage for sequencing. Enzymes like trypsin and chymotrypsin cut proteins at specific sites, creating fragments that can be analyzed. Understanding these cleavage patterns helps researchers piece together the full protein sequence.

Peptide Sequencing Techniques

Edman degradation process

• Method for sequentially identifying amino acids in a peptide from the N-terminus
• Key steps:
  • Phenyl isothiocyanate (PITC) reacts with the N-terminal amino acid under mildly alkaline conditions forms a phenylthiocarbamyl (PTC) derivative
  • Anhydrous acid cleaves the PTC-amino acid from the peptide forming an anilinothiazolinone (ATZ) derivative
  • ATZ derivative is converted to a phenylthiohydantoin (PTH) derivative which can be identified by chromatography or electrophoresis
  • Process is repeated to identify the next amino acid in the sequence
• Limitations:
  • Only works for peptides not full-length proteins
  • Cannot sequence beyond 50-60 amino acids due to inefficiencies in the process
  • N-terminal modifications (acetylation) can block the reaction
  • Cannot identify the C-terminal amino acid
  • Requires a pure peptide sample

Partial hydrolysis for protein sequencing

• Breaks large proteins into smaller overlapping peptide fragments that can be sequenced individually
• Chemical methods:
  • Use dilute acid (6M HCl) or base (1M NaOH) to randomly hydrolyze peptide bonds
  • Resulting peptide fragments are separated and sequenced using techniques like Edman degradation
  • Overlapping sequences are used to reconstruct the full protein sequence
• Enzymatic methods:
  • Use proteases with specific cleavage sites to generate peptide fragments
  • Examples include trypsin (cleaves after Lys and Arg) and chymotrypsin (cleaves after Phe, Tyr, and Trp)
  • Resulting peptide fragments are separated and sequenced using techniques like Edman degradation
  • Overlapping sequences are used to reconstruct the full protein sequence
• Comparison:
  • Chemical methods are less specific and generate more complex mixtures of peptides
  • Enzymatic methods are more specific and generate a more predictable set of peptide fragments
  • Enzymatic methods are often preferred due to their specificity and reproducibility

Enzyme specificity in peptide cleavage

• Trypsin specificity:
  • Cleaves peptide bonds on the C-terminal side of lysine (Lys, K) and arginine (Arg, R) residues
  • Exceptions: cleavage is blocked if Lys or Arg is followed by proline (Pro, P)
  • Example: AKSDARFG → AK | SDAR | FG
• Chymotrypsin specificity:
  • Cleaves peptide bonds on the C-terminal side of phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W) residues
  • Cleavage is more efficient if these residues are followed by a residue with a small side chain (Ala, Gly, Ser)
  • Example: AKFDSYGW → AKF | DSY | GW
• Predicting fragments:
  1. Identify the specific cleavage sites for each enzyme in the protein sequence
  2. Determine the expected peptide fragments based on these cleavage sites
  3. Consider any exceptions or factors that may influence cleavage efficiency

Protein Structure and Sequencing Methods

• Primary structure refers to the linear sequence of amino acids in a polypeptide chain
• Protein sequencing techniques aim to determine the amino acid sequence of a protein
• Mass spectrometry is a powerful tool for analyzing protein structure and sequence
  • Can provide information on peptide fragments and post-translational modifications
  • Often used in conjunction with other sequencing methods for comprehensive analysis