The specific isolation of C-terminal peptides of proteins through a transamination reaction and its advantage for introducing functional groups into the peptide.
A novel method for isolating C-terminal peptides from proteolytic digests of proteins was developed. Proteins were digested with lysyl endopeptidase (LysC) and applied to metal-ion-catalyzed transamination reactions. This reaction enabled the selective conversion of an Nalpha-amino group to a carbonyl group. Subsequent incubation with p-phenylenediisothiocyanate (DITC) glass effectively scavenged the lysine-containing N-terminus and internal peptides. The obtained C-terminal peptide is open to modification with reagents having virtually any type of functionality owing to the reactive alpha-ketocarbonyl group. In this report, 2,4-dinitrophenylhydrazine (DNPH) was used as an example of a nucleophile to the carbonyl group. The isolated C-terminal peptide was modified with DNPH, which exhibited signal enhancement, and was sequenced by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).
Sonomura
Kazuhiro
K
Institute for Protein Research, Osaka University, Suita 565-0871, Japan.
Kuyama
Hiroki
H
Matsuo
Ei-Ichi
E
Tsunasawa
Susumu
S
Nishimura
Osamu
O
D000588:Amines / Q000737:chemistry*; D000595:Amino Acid Sequence; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000588:Amines; D010455:Peptides; D008239:Lysine