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.

Hemorrhagic fever outbreaks such as Ebola are difficult to detect and control because of the lack of low-cost, easily deployable diagnostics and because initial clinical symptoms mimic other endemic diseases such as malaria. Current molecular diagnostic methods such as polymerase chain reaction require trained personnel and laboratory infrastructure, hindering diagnostics at the point of need. Although rapid tests such as lateral flow can be broadly deployed, they are typically not well-suited for differentiating among multiple diseases presenting with similar symptoms. Early detection and control of Ebola outbreaks require simple, easy-to-use assays that can detect and differentiate infection with Ebola virus from other more common febrile diseases. Here, we developed and tested an immunoassay technology that uses surface-enhanced Raman scattering (SERS) tags to simultaneously detect antigens from Ebola, Lassa, and malaria within a single blood sample. Results are provided in <30 min for individual or batched samples. Using 190 clinical samples collected from the 2014 West African Ebola outbreak, along with 163 malaria positives and 233 negative controls, we demonstrated Ebola detection with 90.0% sensitivity and 97.9% specificity and malaria detection with 100.0% sensitivity and 99.6% specificity. These results, along with corresponding live virus and nonhuman primate testing of an Ebola, Lassa, and malaria 3-plex assay, indicate the potential of the SERS technology as an important tool for outbreak detection and clinical triage in low-resource settings.

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