A Mass Spectrometric Approach for Characterization of Amyloid-β Aggregates and Identification of Their Post-Translational Modifications

Abstract

Endogenous amyloid-β (Aβ) oligomeric aggregates have been proposed as toxic agents in Alzheimer's disease (AD). Knowledge of their structures not only may provide insight into the basis of their neurotoxicities but also may reveal new targets for therapeutic drugs and diagnostic tools. However, the low levels of these Aβ oligomers have impeded structural characterization. Evidence suggests that the endogenous oligomers are covalently modified in vivo. In this report, we demonstrate an established mass spectrometry (MS) methodology called precursor ion mapping (PIM) that potentially may be applied to endogenous oligomer characterization. First, we illustrate the use of this PIM technique with a synthetic Aβ(1-40) monomer sample that had been cross-linked with transglutaminase (TGase) and digested with pepsin. From PIM analysis of an Aβ(4-13) MS/MS fragment, precursor ions were identified that corresponded to peptic fragments of three TGase cross-linked species: Aβ(4-19)--(4-19), Aβ(4-19)--(20-34), and Aβ(1-19)--(20-34). Next, we demonstrate the applicability of the PIM technique to an endogenous Aβ sample that had been purified and concentrated by immunoaffinity chromatography. Without pepsin digestion, we successfully identified the full length and C-terminally truncated monomeric Aβ species 1-35 to 1-42, along with select methionine-oxidized counterparts. Because PIM focuses only on a subpopulation of ions, namely the related precursor ions, the resulting spectra are of increased specificity and sensitivity. Therefore, this methodology shows great promise for structural analysis and identification of post-translational modification(s) in endogenous Aβ oligomers.

Protein post‐translational modifications and misfolding: New concepts in heart failure

“…Notably, the last cleavage is catalyzed by γ‐secretase, which can cleave at different sites (38, 40, and 42). Of these, the Aβ42 product showed the highest propensity to form toxic PAOs in vivo . This, along with other examples, points to a role of specific PTMs in triggering amyloid formation .…”

“…A recent example of MS applied to the study of PAOs’ PTMs addressed the role of protein cross‐linking, which can be catalyzed by transglutamminases. In this study Tay et al used a targeted approach combined with in vitro experiments to predict the mass of several peptide fragments originated by cross‐linked peptides . The study shows that targeted approaches can be used to monitor specific PTMs that could be challenging to measure and monitor with proteome‐wide approaches and underscore the importance of targeted approaches in the future of proteomics .…”

Neuronal heparan sulfates promote amyloid pathology by modulating brain amyloid-β clearance and aggregation in Alzheimer’s disease

“…The samples and standards were added to the plates and incubated overnight at 4°C. The Aβ oligomers used as standards were prepared and characterized as previously described (79, 80). The plates were washed three or more times with wash buffer between each step of the assay.…”

Detection of Aβ Monomers and Oligomers: Early Diagnosis of Alzheimer's Disease

“…[96] Under carefullyo ptimized conditions, the oligomerization processa nd the kinetics of in vitro Ab oligomer formation were monitored by the use of the LILBID technique because thisa pproach preserves specific protein-protein interactions.T ay et al also demonstrated that ap recursor ion mapping( PIM)-MS methodology could be appliedt ot he characterization of endogenous Ab oligomers. [97] Hence,M S-based techniques are useful to improve the understandingo fA b oligomerization and to assist in therapeutic treatments for slowing disease progression in patients with AD.…”