Separation of β-Amyloid Tryptic Peptide Species with Isomerized and Racemized l-Aspartic Residues with Ion Mobility in Structures for Lossless Ion Manipulations

Abstract

Accumulation of β-amyloid (Aβ) is one of the hallmarks of Alzheimer’s disease. The deposition of β-amyloid plaques is likely to start years in advance of manifestation of clinical symptoms, although the exact timing is unknown. Over the years, Aβ peptides undergo both post-translational modification and stereoisomerization. Analysis of the resulting stereoisomers is particularly challenging because of their identical elemental composition and similar physicochemical properties. Herein, we have utilized our recently developed structures for lossless ion manipulations ion mobility-mass spectrometry platform (SLIM IM-MS), in conjunction with serpentine ultralong path with extended routing (SUPER), to baseline resolve four distinct sets of Aβ17–28 tryptic peptide epimers on a rapid (~1 s) time scale. We discovered that sodium adduct ions, [M + H + Na]2+, allowed baseline SLIM SUPER IM resolution for all Aβ epimer sets assessed, while such baseline separations were unachievable for their [M + 2H]2+ doubly protonated ions.

Quantification of N-terminal amyloid-β isoforms reveals isomers are the most abundant form of the amyloid-β peptide in sporadic Alzheimer’s disease

“…With the current resolution of R ∼ 50 for our DT-IMS-MS method, it is not possible to rule out any other amino acid (such as Ser) isomerization on this peptide. Future investigations with techniques like SLIM-IMS providing higher resolution ( R > 300) 46 , 47 will lead to better understanding and characterization of other low abundant structural PTMs of Aβ in Alzheimer’s disease brains.…”

“… 41–45 Recently, synthetic tryptic Aβ peptide isomers have been shown to resolve in IMS-MS using structures for lossless ion manipulations (SLIM). 46 , 47 Coupling of online LC to SLIM-IMS demonstrated the potential of LC-IMS-MS in resolving challenging peptide isomers. 47 …”

“… 46 , 47 Coupling of online LC to SLIM-IMS demonstrated the potential of LC-IMS-MS in resolving challenging peptide isomers. 47 …”

Quantitative shotgun proteome analysis by direct infusion

“…Monoclonal cell lines were expanded, frozen down, and validated for PPTC7 knockout via Western blotting for endogenous Pptc7 (Novus, cat # NBP1–90654). The specificity of this antibody was validated using wild type and Pptc7 −/− mouse embryonic fibroblasts derived from a previously generated Pptc7 −/− mouse model 28 .…”

“…Alongside these recent MS advancements, ion mobility has emerged as a promising method to enable gas-phase separations of peptide cations [15][16][17][18][19][20][21][22] . Unlike liquid separations that work on the principle of hydrophobicity, the ion mobility separations sort gas-phase peptide ions based on their charge and shape, which complements separation based on mass/charge achieved by quadrupole mass analyzers.…”