The removal of 2-oxoacyl residues from the N-terminus of peptides and cystatin in non-denaturing conditions.
The N-terminal residue of a protein or peptide may be converted into a 2-oxoacyl group by non-enzymic transamination. This group may then be removed, to obtain the peptide chain shortened by one residue, by treatment with phenylene-1,2-diamine. Hitherto this scission has required a pH of 4-5, but we find that the reaction will proceed well at pH 7 in the presence of concentrated phosphate buffer. We describe a method using reverse-phase HPLC for determining the extent of scission in model peptides; this method also allows products to be isolated and identified. The new scission conditions have been tested by removing the N-terminal residue from cystatin, an inhibitor of cysteine peptidases; electrospray mass spectrometry was used to assess how this protein reacted.
Stevens
J
J
Department of Biochemistry, University of Cambridge, UK.
Dixon
H B
HB
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D015891:Cystatins / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D010504:Periodic Acid; D010655:Phenylenediamines; D011489:Protein Denaturation; D000637:Transaminases / Q000737:chemistry
D015891:Cystatins; D010455:Peptides; D010655:Phenylenediamines; D010504:Periodic Acid; C009288:metaperiodate; D000637:Transaminases