title: A novel derivatization method with 5-bromonicotinic acid N-hydroxysuccinimide for determination of the amino acid sequences of peptides.
abstract: We have developed a novel method that effectively identifies the N-terminal product ions produced in the tandem mass spectrometry (MS/MS) analysis of peptides done in conjunction with the specific derivatization of the N-terminal amino group using 5-bromonicotinic acid N-hydroxysuccinimide ester (BrNA-NHS). Electrospray ionization with low-energy collision-induced dissociation (CID) MS/MS clearly differentiated the N-terminal product ions labeled with the 5-bromonicotinyl group from other ions, on the basis of the appearance of CID peaks with a doublet pattern characteristically separated by 2 mass units produced by the equal natural abundances of 79Br and 81Br. The tracing of a series of these bromine-containing product ions allows the easy amino acid sequencing of peptides. Using Gln-Arg-Leu-Gln-Ser-Asn-Gln-Leu-Lys as the test peptide, we found that within 30 minutes at pH 6.5 and 37 degrees C its alpha-amino group was completely acylated with BrNA-NHS (peptide: BrNA-NHS = 1:40; mol/mol). The epsilon-amino group of the C-terminal lysine residue was less likely to be acylated under these conditions, being only partly modified (about 20%). This suggests the possibility of keeping the epsilon-amino group free from acylation. The method was successfully applied to the determination of the amino acid sequences of peptides from porcine kidney aminoacylase I produced by digestion with lysyl endopeptidase and with Staphylococus aureus V8 protease.
authors.affiliation: Biotechnology Research Laboratories, Takara Shuzo Co., Ltd., Otsu, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Miyagi
authors.forename: M
authors.initials: M
authors.lastname: Nakao
authors.forename: T
authors.initials: T
authors.lastname: Nakazawa
authors.forename: I
authors.initials: I
authors.lastname: Kato
authors.forename: S
authors.initials: S
authors.lastname: Tsunasawa
mesh_terms: D000595:Amino Acid Sequence; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009525:Niacin / Q000031:analogs & derivatives* / Q000737:chemistry; D010455:Peptides / Q000032:analysis*; D017421:Sequence Analysis; D013388:Succinimides / Q000737:chemistry*
chemical_list: C112927:5-bromonicotinic acid N-hydroxysuccinimide ester; D007202:Indicators and Reagents; D010455:Peptides; D013388:Succinimides; D009525:Niacin