Enhanced ion mobility resolution of Abeta isomers from human brain using high-resolution demultiplexing software

Isomerization of aspartic acid (Asp) residues in long-lived proteins is a key feature associated with neurodegenerative proteinopathies such as Alzheimer's disease (AD). Recently, using ultra high-performance liquid chromatography (UHPLC) coupled with drift tube ion mobility mass spectrometry (DTIMS-MS), we documented the extensive Asp isomerization in amyloid-beta (Aβ) peptides depositing in the extracellular cortical plaques (senile plaques) of the AD brain. Aβ_1-15 was estimated to be ~ 85% isomerized, while Aβ_4-15 another major constituent of these senile plaques was ~ 50% isomerized in AD brain. Low resolution on the standard demultiplexed ion mobility resulted in poor separation of these N-truncated Aβ isomers in the ion mobility domain. Here, using the same ion multiplexed dataset, we applied new post-acquisition data reconstruction technique, high-resolution demultiplexing (HRdm), to improve the resolution of these Aβ isomers in the ion mobility dimension. We demonstrate that for the complex proteomic AD brain digests, HRdm could successfully resolve three out of four major Asp isomers of Aβ_1-15. For Aβ_2-15 and Aβ_4-15, the significant resolution enhancement in the HRdm data resulted in baseline peak separation of the respective Asp isomers. An analysis of two-peak resolution (Rpp) and peak-to-peak separation (ΔP) indicated twofold enhancement for the Asp-isomerized Aβ species. HRdm performed with an effective resolving power (Rp) of between 150 and 160 for the highest deconvolution settings in comparison to ~ 40 to 65 in the standard settings. These major resolution improvements in the ion mobility domain for the endogenous Aβ isomers demonstrate the feasibility of in situ measurement of peptide isomers and their role in the mechanism of amyloid plaque formation in AD.