This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Deamidation of asparaginyl residues in proteins is a post-translational modification resulting in a mixture of aspartyl and isoaspartyl residues and thought to be responsible for the inactivation and misfolding of proteins. Of the two isomeric products, isoaspartyl is thought to be the most damaging to protein activity because the primary structure is shifted by the insertion of a methylene group into the protein backbone thus making differentiation of the two forms important. ECD (electron capture dissociation) has been shown to differentiate the two forms in synthetic peptides based on characteristic fragment ions of each form. Data presented here shows that these ECD fragment ions are reproducible in tryptic peptides from a deamidated protein proving the method?s applicability to protein analysis.ECD analysis was performed on a home built qQq-FTMS (Fourier transform mass spectrometer with mass filtering front-end quadrupoles and CAD cell) equipped with a nano-spray source and 7T actively shielded magnet. For each experiment, the multiply charged precursor ions were isolated in Q1, externally accumulated in Q2 and then transmitted to the ICR cell for ECD and subsequent detection. A tryptic fragment of cytochrome C (H-TGPNLHGLFGR-OH, m/z = 584.8153, 2+) was fully deamidated overnight at 80 C and pH 12 indicated by a mass shift of approximately 1 dalton. Calmodulin was incubated at 37 C and pH 8 for two weeks then digested by tryspin and a tryptic peptide (H-VFDKDGNGYISAAELR-OH, m/z = 585.6290, 3+) was shown to be completely deamidated.The ECD spectrum of the cytochrome C deamidated tryptic peptide showed all z? ions within the range of detection although only four c ions were detected (c7-c10) and were in general of lower abundance due to the N-terminal arginine residue. A peak corresponding to the z8-57 (1 ppm) fragment indicated the presence of the isoaspartyl residue. No complimentary fragment ion (c?+58) was found in the spectrum. The deficiency of this ion is most likely due to the position of the arginine residue in concurrence with the fact that the diagnostic isoaspartyl ions are typically of lower abundance than the c/z? series ion. A peak corresponding to the neutral loss of 60 daltons, the loss of the aspartic acid side chain from the reduced precursor ion, was not found indicating that the aspartyl product was of much lower abundance than the isoaspartyl form. The ECD spectrum of the calmodulin tryptic peptide showed 12 c and 12 z? ions, all of which are in similar abundances most likely due to the N-terminal arginine residue and the lysine residue close to the C-terminus. Both the c7?+58 and z8-57 ions were found (1 ppm) indicating the presence of the isoaspartyl residue substituted for the arginine residue. The peak corresponding to the loss of the aspartic acid side chain from the reduced precursor ion for this peptide was of considerable abundance but cannot provide unambiguous evidence of the aspartyl form because of the two aspartyl residues in the peptide.The results above show that the isoaspartyl product from deamidation of asparaginyl residues in peptides and proteins can be detected based on the presence of the c?+58 and z8-57 diagnostic ions. Furthermore, the relative abundance of these diagnostic ions has been shown to be linear with their mole ratio so that quantitation of the asp/isoasp ratio in a site-specific manner is possible. Further testing is necessary to determine if this extends to whole proteins and is ongoing.
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