A method for terminus proteomics: selective isolation and labeling of N-terminal peptide from protein through transamination reaction.
A novel method for selectively labeling and isolating N-terminal peptide from protein has been developed. An N(alpha)-amino group of protein was converted to a carbonyl group through transamination reaction and the resulting carbonyl group was modified with O-(4-nitrobenzyl)hydroxylamine (NBHA). After proteolytic digestion using Grifola frondosa metalloendopeptidase (LysN), the modified N-terminal peptide remained unbound in the following treatment using amino-reactive p-phenylenediisothiocyanate (DITC) glass, whereas peptides other than the N-terminal peptide were effectively scavenged from the supernatant solution. The modified N-terminal peptide was thus successfully isolated and sequenced by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) analysis.
Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan.
Kazuhiro
K
Sonomura
Hiroki
H
Kuyama
Ei-ichi
E
Matsuo
Susumu
S
Tsunasawa
Osamu
O
Nishimura
D001487:Basidiomycota / Q000201:enzymology; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification*; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling; D053719:Tandem Mass Spectrometry
D010455:Peptides; D011506:Proteins; D008666:Metalloendopeptidases