C-terminal ladder sequencing of peptides using an alternative nucleophile in carboxypeptidase Y digests
2006 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 357, no 2, 167-172 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2006. Vol. 357, no 2, 167-172 p.
2-Pyridylmethylamine; MALDI; Matrix-assisted laser desorption/ionization mass spectrometry; Transpeptidation
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:kth:diva-7106DOI: 10.1016/j.ab.2006.07.025ISI: 000241077800002ScopusID: 2-s2.0-33748784378OAI: oai:DiVA.org:kth-7106DiVA: diva2:12019