title: Selective isolation and identification of N-terminal blocked peptides from tryptic protein digests.
abstract: A method for the easy isolation and direct sequencing of N-terminally blocked peptide in proteins refractory to N-terminal sequencing was developed. It is based essentially on tandem enzymatic treatments of the protein with trypsin and carboxypeptidase B, and selective isolation of the Nalpha-blocked peptide using ion-exchange chromatography. The chromatographic step was optimized for picomole amounts of sample and very short elution times by placing a thin layer of the resin over the membrane of an ultrafiltration tube. The isolated fraction can be analyzed directly using MALDI or ESI mass spectrometry. The method was applied to several recombinant and natural N-terminal acetylated proteins. A critical discussion on the intrinsic limitations of the method is also given.
authors.affiliation: Center for Genetic Engineering and Biotechnology, Havana, Cuba.
authors.forename: L
authors.initials: L
authors.lastname: Betancourt
authors.forename: V
authors.initials: V
authors.lastname: Besada
authors.forename: L J
authors.initials: LJ
authors.lastname: González
authors.forename: V
authors.initials: V
authors.lastname: Morera
authors.forename: G
authors.initials: G
authors.lastname: Padrón
authors.forename: T
authors.initials: T
authors.lastname: Takao
authors.forename: Y
authors.initials: Y
authors.lastname: Shimonishi
mesh_terms: D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification*; D010449:Peptide Mapping; D011506:Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry*
chemical_list: D010446:Peptide Fragments; D011506:Proteins; D014357:Trypsin