Selective isolation and identification of N-terminal blocked peptides fromtryptic protein digests

A method for the easy isolation and direct sequencing of N-terminally block ed peptide in proteins refractory to N-terminal sequencing was developed. I t is based essentially on tandem enzymatic treatments of the protein with t rypsin and carboxypeptidase B, and selective isolation of the N-alpha-block ed peptide using ion-exchange chromatography. The chromatographic step was optimized for picomole amounts of sample and very short elution times by pl acing a thin layer of the resin over the membrane of an ultrafiltration tub e. The isolated fraction can be analyzed directly using MALDI or ESI mass s pectrometry. The method was applied to several recombinant and natural N-te rminal acetylated proteins. A critical discussion on the intrinsic limitati ons of the method is also given.