A NOVEL DERIVATIZATION METHOD WITH 5-BROMONICOTINIC ACID N-HYDROXYSUCCINIMIDE FOR DETERMINATION OF THE AMINO-ACID-SEQUENCES OF PEPTIDES

We have developed a novel method that effectively identifies the N-ter minal product ions produced in the tandem mass spectrometry (MS/MS) an alysis of peptides done in conjunction with the specific derivatizatio n of the N-terminal amino group using 5-bromonicotinic acid N-hydroxys uccinimide ester (BrNA-NHS). Electrospray ionization with low-energy c ollision-induced dissociation (CID) MS/MS clearly differentiated the N -terminal product ions labeled with the 5-bromonicotinyl group from ot her ions, on the basis of the appearance of CID peaks with a doublet p attern characteristically separated by 2 mass units produced by the eq ual natural abundances of Br-79 and Br-81. Th, tracing of a series of these bromine-containing product ions allows the easy amino acid seque ncing of peptides. Using Gln-Arg-Leu-Gln-Ser-Asn-Gln-Leu-Lys as the te st 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 VS protease. (C ) 1998 John Wiley & Sons, Ltd.