Identification and quantitation of tetrapeptide deamidation products by mass spectrometry

A method to quantify asparagine (Asn), aspartate (Asp) and isoaspartate (isoAsp) residues in small peptides by fast atom bombardment mass spectrometry (FAB-MS) was developed. Discrimination of isoAsp from Asp residues was accomplished by selective derivatization of isoAsp residues in acetic anhydride, D2O and pyridine. Deuteration occurred at any carbon adjacent to a free alpha-carboxyl group, through a transient oxazalone intermediate, allowing the isoAsp side chain and the C-terminus to incorporate deuterium. Thus, isoAsp-containing peptides incorporate one more deuterium than peptides with Asp and two more than Asn peptides. FAB CID-MS spectra of the Asn tetrapeptide, Thr-Asn-Ser-Tyr, were used to confirm the position of deuteration to the C-terminal residue. FAB and FAB CID-MS spectra demonstrated that the 1 amu shift in mass was not caused by derivatization induced deamidation of the Asn residue. FAB-MS spectra of deuterated peptide standards and mixtures containing deamidation products were obtained over the molecular ion region and deconvoluted using non-deuterated control spectra. Deuterium incorporation values for the Asn, Asp and iosAsp containing peptide standards were 80% mono-deuterated peptide, 95% mono-deuterated peptide and 63% di-deuterated peptide, respectively. IsoAsp to Asp ratios in an unknown mixture were obtained by a least-squares minimization of the difference between the unknown deuterated mixture and the isotopic envelopes from the deuterated standards. The mixture was found to contain 85% isoAsp peptide by FAB-MS, which agreed well with 81% isoAsp peptide when assayed by reversed-phase LC.