Single‐Molecule Detection of Isoform‐Specific Tau Phosphorylation

Abstract

AbstractBackgroundPost‐translational modifications (PTMs) contribute to the vast diversity of proteoforms that drive biological processes. Currently, very little is known about the molecular heterogeneity of proteoforms. This gap can be dominantly attributed to a lack of methods to analyze PTMs on intact single protein molecules on a proteome‐scale.MethodHere, we introduce an antibody‐based single‐molecule analysis platform that includes novel biochemistry for single‐protein molecule immobilization, instrumentation for highly sensitive detection, and computation for data interpretation. We use this platform to study Tau proteoforms. Aberrant PTMs on Tau generate a wide diversity of proteoforms that lead to Tau misfolding and aggregation and are associated with neurodegenerative disorders collectively referred to as Tauopathies.ResultFirst, we validate the platform by measuring defined mixtures of recombinant tau proteins. Next, we use the platform to examine tau protein enriched from human induced pluripotent stem cell (iPSC)‐derived neurons and tau‐expressing cell lines. As PTMs can be assigned to individual protein molecules, the platform is able to reveal the molecular heterogeneity of tau proteoforms that is missed by both bulk measurements and by peptide‐centric proteomics approaches.ConclusionUltimately, quantitative analysis of proteoform molecular heterogeneity will lead to a more detailed view of the tau proteoform landscape towards advancing precision medicine of tauopathies, such as Alzheimer’s disease.