A method for N-terminal de novo sequence analysis of proteins by matrix-assisted laser desorption/ionization mass spectrometry.
A novel method for isolation and de novo sequencing of N-terminal peptides from proteins is described. The method presented here combines selective chemical tagging using succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-Ac-OSu) at the N(alpha)-amino group of peptides after digestion by metalloendopeptidase (from Grifola frondosa) and selective capture procedures using p-phenylenediisothiocyanate resin, by which the N-terminal peptide can be isolated, whether or not it is N-terminally blocked. The isolated N-terminal peptide modified N-terminally with TMPP-Ac-OSu reagent produces a simple fragmentation pattern under tandem mass spectrometric analysis to significantly facilitate sequencing.
Kuyama
Hiroki
H
Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan. kuyama@protein.osaka-u.ac.jp
Sonomura
Kazuhiro
K
Nishimura
Osamu
O
Tsunasawa
Susumu
S
D000818:Animals; D024402:Carbonic Anhydrase II / Q000032:analysis / Q000737:chemistry; D006454:Hemoglobins / Q000032:analysis / Q000737:chemistry; D006801:Humans; D007768:Lactalbumin / Q000032:analysis / Q000737:chemistry; D010446:Peptide Fragments / Q000032:analysis* / Q000302:isolation & purification; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D020539:Sequence Analysis, Protein / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D006454:Hemoglobins; D010446:Peptide Fragments; D011506:Proteins; D007768:Lactalbumin; D024402:Carbonic Anhydrase II