Deamidation: Differentiation of aspartyl from isoaspartyl products in peptides by electron capture dissociation

Abstract

Deamidation of asparaginyl and isomerization of aspartyl residues in proteins proceed through a succinimide intermediate producing a mixture of aspartyl and isoaspartyl residues. Isoaspartic acid is an isomer of aspartic acid with the C ␤ incorporated into the backbone, thus increasing the length of the protein backbone by one methylene unit. This post-translation modification is suspected to contribute to the aging of proteins and to protein folding disorders such as Alzheimer's disease, so that differentiating the two isomers becomes important. This manuscript reports that distinguishing aspartyl from isoaspartyl residues in peptides has been accomplished by electron capture dissociation (ECD) using a Fourier transform mass spectrometer (FTMS). Model peptides with aspartyl residues and their isoaspartyl analogs were examined and unique peaks corresponding to c n ·+58 and z ᐉ−n −57 fragment ions (n, position of Asp; ᐉ, total number of amino acids in the peptide) were found only in the spectra of the peptides with isoaspartyl residues. The proposed fragmentation mechanism involves cleavage of the C ␣ -C ␤ backbone bond, therefore splitting the isoaspartyl residue between the two fragments. Also, a complementary feature observed specific to aspartyl residues was the neutral loss of the aspartic acid side chain from the charge reduced species. CAD spectra of the peptides from the same instrument demonstrated the improved method because previously published CAD methods rely on the comparison to the spectra of standards with aspartyl residues. The potential use of the top-down approach to detect and resolve products from the deamidation of asparaginyl and isomerization of aspartyl residues is discussed.

Use of 18O labels to monitor deamidation during protein and peptide sample processing

“…In collisionally activated dissociation (CAD), the two isomers have different, identifiable side-chain fragmentation patterns for the N-and C-terminal ions [22,24,25]. In electron capture dissociation (ECD) [26 -29] and electron-transfer dissociation (ETD) [30,31], their fragmentations also produce different diagnostic ions: the [(M ϩ nH) (nϪ1)ϩ· Ϫ 60] fragment ion for the detection of the Asp, and the c · ϩ 58 and z Ϫ 57 ions for the detection and location of the isoAsp [32][33][34][35]. However, ECD of intact proteins is often inefficient because the number of available fragmentation channels is large and the resulting fragments frequently remain bound by noncovalent interactions and are thus undetectable.…”

Differentiation of aspartic and isoaspartic acids using electron transfer dissociation

“…Because isoaspartic acid does not have an nOc ␣ bond, it was postulated that the ECD spectra of aspartic acid-containing peptides versus isoaspartic acid containing peptides would be substantially different, allowing facile differentiation of the two residues, a hypothesis that was borne out by experiment [11]. In these experiments, the c ␣ Oc ␤ bond was cleaved, which generated a diagnostic side-chain cleavage for aspartic acid residues, at M-60 Da and two diagnostic backbone cleavages at c n ϩ 57 (or c n • ϩ 58) and z m Ϫ 57 (where m ϭ peptide length Ϫ n).…”

Characterization of a new qQq-FTICR mass spectrometer for post-translational modification analysis and top-down tandem mass spectrometry of whole proteins

“…High resolving power (100,000 FWHM) and mass accuracy of ϳ1 ppm could be readily achieved when an internal calibration of the spectra was performed (in this case the y 10 ϩ and y 10 2ϩ ions were used to internally calibrate the spectrum). Although in typical MS 2 spectra resolving the ϪH 2 O/ϪNH 3 species is not biologically interesting, it is important to note that this mass shift is the same shift observed on deamidation of asparagine or glutamine°residues° [45];°this°post-translational°modifi-cation is linked to protein aging, protein folding disorders, and protein degradation.…”