Distribution of isomerized and racemized amyloid β isoforms in sporadic Alzheimer’s disease using ion‐mobility mass spectrometry: Molecular and cell biology: Importance of Abeta/amyloid in brain changes

Background Extracellular amyloid plaques and intracellular neurofibrillary tangles are the pathological hallmarks of Alzheimer’s Disease (AD). It takes on average 19 years for amyloid β (Aβ) peptides to deposit as insoluble plaques from onset to clinical dementia symptoms in AD. Such long‐lived nature of amyloidosis predisposes the polypeptide chains to undergo multiple spontaneous and non‐enzymatic post‐translational modifications (PTMs), which can render them resistant to normal cellular proteolysis mechanisms. Several biochemical approaches have estimated very high degree of isomerization and racemization of Asp and Ser residues in Aβ purified from the insoluble plaques. This goes along with a sequential loss of N‐terminal amino acids. However, comprehensive characterisation of these isomers as well as their quantitative estimation is yet to be done. Method Freshly frozen post‐mortem frontal temporal cortex samples from non‐demented brains (n=10) and AD brains (n=11) were homogenised and fractionated into distinct soluble, vesicular, membranous and insoluble/aggregated biological pools of Aβ. The Aβ rich pools were enzymatically digested and separated on UHPLC coupled to ESI‐drift tube ion mobility (DTIM) mass spectrometer (Agilent Technologies, Santa Clara, USA). Stable isotope labeled peptides (SIS) were spiked in these brain homogenates to quantify the amount of these isomers/racemers in the different fractionated biochemical pools of the temporal cortex grey matter of human AD and control brains. Result We have characterised and quantified the most relevant Aβ isomers (stereoisomers of the N‐terminus) from AD brains by using their SIS peptides. Isomer separation was achieved by LC and their collisional cross section ( DT CCS N2 ) values. Significant isomerization was observed in AD compared to controls brains. Moreover, the amount of these isomerized and racemized peptides differed from lower levels in the soluble/peripheral membranous to higher levels in the insoluble/aggregated debris in AD brain. These findings have implications in Aβ neurotoxicity, oligomerization, structures of amyloid fibrils present in the AD brain. Conclusion The results show that isomerized/racemized Aβ peptides are intricately associated with the accumulation and deposition of amyloid in the brain, allowing distinct differentiation between old plaques and freshly generated Aβ. Specifically targeting these long‐lived isomers for enhanced clearance from the CNS would be crucial in developing any therapeutics targeting Aβ.