BCH210H1 Lecture 11: Analysis of Proteins—Chromatography, Sequences- Sequences

• School: UTSG
• Department: Biochemistry
• Course: BCH210H1
• Professor: Stavroula Andreopoulos

Document Summary

Edman degradation removes the n- terminal amino acid: chromophore used is phenylisothiocyanate, attach it to polypeptide at n- terminus, nucleophilic reaction attacks first n-terminal residue and chromophore attaches to it. Protein sequencing: determination of sequence from c-terminal end (last amino acid, typically enzymatic in nature, method use carboxypeptidase enzyme that cleaves amino acids one at a time from the c- terminus, detect. Peptide fragment approach: edman degradation and carboxypeptidase methods may be limited by length of sequence and modification of proteins, phosphorylated serine. Edman degradation would not work: method break protein sequence into smaller fragments and then run each fragment through edman degradation separately. Protein cleavage methods: subtilisin can cleave many peptide bonds (broad specificity, trypsin, chymotrypsin enzyme cleaves to carboxyl side of basic residues (lys, arg) enzyme cleaves to carboxyl side of aromatic residues (trp, Overlap of peptide fragments: aaar, faaak, laaamaaaa, aaarf, aaaklaaamaaaa, aaarfaaaklaaam, aaaa, these peptides can be used to reassemble sequence.