To explore the mechanism of electron capture dissociation (ECD) of linear peptides, a set of 16-mer peptides were synthesized with deuterium labeled on the ␣-carbon position of four glycines. The ECD spectra of these peptides showed that such peptides exhibit a preference for the radical to migrate to the ␣-carbon position on glycine via hydrogen (or deuterium) abstraction before the final cleavage and generation of the detected product ions. The data show c-type fragment ions, ions corresponding to the radical cation of the c-type fragments, c·, and they also show c·-1 peaks in the deuterated peptides only. The presence of the c·-1 peaks is best explained by radical-mediated scrambling of the deuterium atoms in the long-lived, metastable, radical intermediate complex formed by initial electron capture, followed by dissociation of the complex. These data suggest the presence of at least two mechanisms, one slow, one fast. The abundance of H· and ϪCO losses from the precursor ion changed upon deuterium labeling indicating the presence of a kinetic isotope effect, which suggests that the values reported here represent an underestimation of radical migration and H/D scrambling in the observed fragments. (J Am Soc Mass Spectrom 2006, 17, 576 -585)

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.

BackgroundThere is current expansion of newborn screening (NBS) programs to include lysosomal storage disorders because of the availability of treatments that produce an optimal clinical outcome when started early in life.ObjectiveTo evaluate the performance of a multiplex-tandem mass spectrometry (MS/MS) enzymatic activity assay of 6 lysosomal enzymes in a NBS laboratory for the identification of newborns at risk for developing Pompe, Mucopolysaccharidosis-I (MPS-I), Fabry, Gaucher, Niemann Pick-A/B, and Krabbe diseases.Methods and ResultsEnzyme activities (acid α-glucosidase (GAA), galactocerebrosidase (GALC), glucocerebrosidase (GBA), α-galactosidase A (GLA), α-iduronidase (IDUA) and sphingomyeline phosphodiesterase-1 (SMPD-1)) were measured on ~43,000 de-identified dried blood spot (DBS) punches, and screen positive samples were submitted for DNA sequencing to obtain genotype confirmation of disease risk. The 6-plex assay was efficiently performed in the Washington state NBS laboratory by a single laboratory technician at the bench using a single MS/MS instrument. The number of screen positive samples per 100,000 newborns were as follows: GAA (4.5), IDUA (13.6), GLA (18.2), SMPD1 (11.4), GBA (6.8), and GALC (25.0).DiscussionA 6-plex MS/MS assay for 6 lysosomal enzymes can be successfully performed in a NBS laboratory. The analytical ranges (enzyme-dependent assay response for the quality control HIGH sample divided by that for all enzyme-independent processes) for the 6-enzymes with the MS/MS is 5- to 15-fold higher than comparable fluorimetric assays using 4-methylumbelliferyl substrates. The rate of screen positive detection is consistently lower for the MS/MS assay compared to the fluorimetric assay using a digital microfluidics platform.

Electron-transfer dissociation allows differentiation of isoaspartic acid and aspartic acid residues using the same c ϩ 57 and z Ϫ 57 peaks that were previously observed with electron capture dissociation. These peaks clearly define both the presence and the position of isoaspartic acid residues and they are relatively abundant. The lower resolution of the ion trap instrument makes detection of the aspartic acid residue's diagnostic peak difficult because of interference with side-chain fragment ions from arginine residues, but the aspartic acid residues are still clearly observed in the backbone cleavages and can be inferred from the absence of the isoaspartic acid diagnostic ions. (J Am Soc Mass Spectrom 2006, 17, 15-19) © 2005 American Society for Mass Spectrometry N onenzymatic deamidation of asparagine, and at a much lower rate glutamine, residues in proteins occurs spontaneously over time with reaction half-lives that range from ϳ1 day to Ͼ3 years, depending on neighboring amino acid residues and on the higher order folding structure of the protein [1]. Deamidation typically proceeds via SN 2 attack by the backbone amide on the side-chain carbonyl to displace ammonia forming a cyclic succinimide intermediate (Scheme 1) [2]. This intermediate is unstable in aqueous solutions, and because of the symmetry around the nitrogen in the ring, hydration of the succinimide results in formation of a mixture of aspartic acid and isoaspartic acid. In simple peptide studies, the branching ratio of this experiment is usually ϳ3:1 in favor of the isoaspartic acid residue [3]; however, this ratio is unlikely to hold in whole proteins because of conformational constraints.This exchange has two large implications on protein tertiary structure. First, it exchanges a neutral or slightly basic residue for an acidic one-thus changing the charge and hydrogen bonding structure. Second, if an isoaspartic acid residue is generated, an additional methylene unit is placed in the protein backbone, pushing the adjacent carbonyl carbon and ␣-carbons apart by an additional ϳ1-1.5 Å. In both cases, the local environment of the initial asparagine/glutamine is disrupted, which could have dramatic impact on the folding structure of the protein. This nonenzymatic posttranslational modification is important to protein folding studies, including antibody-based therapeutics, which could lose activity or generate unexpected side activities because of a change in their tertiary structure. Furthermore, deamidation is commonly considered one of the fundamental modes by which protein aging is controlled in vivo as evidenced by the existence and importance of a repair enzyme [4,5].Detection of deamidation in peptides and proteins is relatively easy using gel electrophoresis because of the pI change of the protein or using mass spectrometry because of the ϩ0.984 Da mass shift. However, differentiation of the two products, aspartic acid and isoaspartic acid residues, is much harder, because the two isomeric residues are highly similar in reactivity. The ...

The relative abundances of fragment ions in electron capture dissociation (ECD) are often greatly affected by the secondary and tertiary structures of the precursor ion, and have been used to derive the gas-phase conformations of the protein ions. In this study, it is found that resonance ejection of the charge reduced molecular ion during ECD resulted in significant changes in many fragment ion populations. The ratio of the ion peak intensities in the double resonance (DR)-ECD to that in the normal ECD experiment can be used to calculate the lifetime of the radical intermediates from which these fragments are derived. These lifetimes are often in the ms range, a time sufficiently long to facilitate multiple free radical rearrangements. These ratios correlate with the intramolecular noncovalent interactions in the precursor ion, and can be used to deduce information about the gas-phase conformation of peptide ions. DR-ECD experiments can also provide valuable information on ECD mechanisms, such as the importance of secondary electron capture and the origin of c·/z ions. (J Am Soc Mass