title: Selective isolation of N-blocked peptide by combining AspN digestion, transamination, and tosylhydrazine glass treatment.
abstract: Many eukaryotic proteins are blocked at the α-amino group of their N-terminal with various modifications, thereby making it difficult to determine their N-terminal sequence by protein sequencer. We propose a novel method for selectively isolating the blocked N-terminal peptide from the peptide mixture generated by endoproteinase AspN digestion of N-blocked protein. This method is based on removal of all peptides other than the N-terminal one (non-N-terminal peptides) through their carbonyl group introduced by a chemical transamination reaction. The transamination reaction converts the free α-amino group of the non-N-terminal peptides to a carbonyl group, whereas the blocked N-terminal peptide, which lacks only the free α-amino group, remains unchanged. Silica functionalized with the tosylhydrazino group effectively captures non-N-terminal peptides through their carbonyl group; thus, the blocked N-terminal peptide is selectively recovered in the supernatant. This method was applied to several model proteins, and their N-terminal peptides were successfully isolated and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Furthermore, the method was extended to N-terminal analysis of N-free protein by artificially blocking the free α-amino group of its N-terminal with N-succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl) phosphonium bromide reagent.
authors.affiliation: Institute for Protein Research, Osaka University, Osaka 565-0871, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Sonomura
authors.forename: Hiroki
authors.initials: H
authors.lastname: Kuyama
authors.forename: Ei-Ichi
authors.initials: E
authors.lastname: Matsuo
authors.forename: Susumu
authors.initials: S
authors.lastname: Tsunasawa
authors.forename: Shiroh
authors.initials: S
authors.lastname: Futaki
authors.forename: Osamu
authors.initials: O
authors.lastname: Nishimura
mesh_terms: D000586:Amination; D004063:Digestion; D016326:Extracellular Matrix Proteins / Q000737:chemistry*; D007202:Indicators and Reagents / Q000302:isolation & purification; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification*; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D000637:Transaminases / Q000737:chemistry
chemical_list: C439889:ASPN protein, human; D016326:Extracellular Matrix Proteins; D007202:Indicators and Reagents; D010455:Peptides; D011506:Proteins; D000637:Transaminases