Analysis of Tau heterogeneity and proteome‐wide changes in Alzheimer’s Disease at the single‐molecule level

Abstract

AbstractBackgroundThe molecular heterogeneity of Tau is a critical contributor to Alzheimer’s disease (AD) progression and pathology. Currently, very little is known about the prevalence or impact of the diverse collection of Tau proteoforms. Additionally, individual proteoforms may have different interactions or frequencies within the broader proteome. Here, we detected and analyzed Tau proteoforms in the context of the broader proteome in model systems using a novel single‐molecule proteomic analysis platform with future applicability in AD samples.MethodWe used a single‐molecule proteomic analysis platform that leverages a set of target isoform specific and target PTM specific affinity reagents combined with novel instrumentation, single‐molecule biochemistry, and machine learning bioinformatics to enable deep proteoform and broad proteome analysis. In this study, we first evaluated the proteoform distribution of Tau using commercially available antibody reagents. Proteoform heterogeneity was compared (specifically splicing and phosphorylation variants) in both healthy and diseased samples. Then, total protein counts were assessed across the proteome using Protein Identification by Short‐epitope Mapping (PrISM), which leverages proprietary multi‐affinity probes designed to recognize short epitopes and a machine learning algorithm that decodes binding of hundreds of multi‐affinity probes into protein quantifications.ResultThe approach was evaluated by first measuring defined mixtures of recombinant Tau proteins. Next, we examined Tau enriched from induced pluripotent stem cell (iPSC)‐derived neurons and tau‐expressing cell lines. The platform revealed the molecular heterogeneity of Tau proteoforms missed by bulk measurements and peptide‐centric proteomics approaches. The proteoform data was then supplemented with broader proteome analysis in order to identify proteome‐wide changes – differences in proteins and pathways due to or in combination with specific Tau proteoforms and disease.ConclusionUnderstanding the biology of complex diseases like AD and other Tauopathies depends on understanding both the specific Tau proteoforms that exist at baseline and in disease as well as the pathways and processes impacted in the proteome. Single‐molecule tools that can analyze proteoforms in depth and the proteome broadly and link molecular signatures to disease states are essential. These tools will enable improved biomarkers, improved understanding of disease, and improved therapies for Alzheimer’s disease in the future.