title: C-terminal ladder sequencing of peptides using an alternative nucleophile in carboxypeptidase Y digests.
abstract: A method for improved sequence coverage in C-terminal sequencing of peptides, based on carboxypeptidase digestion, is described. In conventional carboxypeptidase digestions, the peptide substrate is usually extensively degraded and a full amino acid sequence cannot be obtained due to the lack of a complete peptide ladder. In the presented method, a protecting group is introduced at the C terminus of a fraction of the peptide fragments formed in the digest, and thereby further degradation of the C-terminally modified peptides are slowed down. The protecting group was attached to the C-terminal amino acid through a carboxypeptidase-catalyzed reaction with an alternative nucleophile, 2-pyridylmethylamine, added to the aqueous digestion buffer. Six peptides were digested by carboxypeptidase Y with and without 2-pyridylmethylamine present in the digest buffer, and the resulting fragments subsequently were analyzed with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Comparison of the two digestion methods showed that the probability of successful ladder sequencing increased, by more than 50% using 2-pyridylmethylamine as a competing nucleophile in carboxypeptidase Y digests.
authors.affiliation: Department of Biochemistry, KTH School of Biotechnology, AlbaNova University Center, SE 106 91 Stockholm, Sweden.
authors.forename: Anders
authors.initials: A
authors.lastname: Hamberg
authors.forename: Martin
authors.initials: M
authors.lastname: Kempka
authors.forename: Johan
authors.initials: J
authors.lastname: Sjödahl
authors.forename: Johan
authors.initials: J
authors.lastname: Roeraade
authors.forename: Karl
authors.initials: K
authors.lastname: Hult
mesh_terms: D043402:Cathepsin A / Q000378:metabolism*; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D020539:Sequence Analysis, Protein / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D010446:Peptide Fragments; D010455:Peptides; D043402:Cathepsin A