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A novel derivatization method with 5‐bromonicotinic acid N‐hydroxysuccinimide for determination of the amino acid sequences of peptides
Author(s) -
Miyagi Masaru,
Nakao Masayuki,
Nakazawa Takashi,
Kato Ikunoshin,
Tsunasawa Susumu
Publication year - 1998
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/(sici)1097-0231(19980529)12:10<603::aid-rcm204>3.0.co;2-0
Subject(s) - chemistry , peptide , derivatization , chromatography , amino acid , electrospray ionization , mass spectrometry , tandem mass spectrometry , lysine , peptide sequence , residue (chemistry) , acylation , biochemistry , gene , catalysis
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 79 Br and 81 Br. 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 °C its α‐amino group was completely acylated with BrNA‐NHS (peptide: BrNA‐NHS = 1:40; mol/mol). The ϵ‐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 ϵ‐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. © 1998 John Wiley & Sons, Ltd.