Single-molecule FRET enables the detection of site-specific α-synuclein serine phosphorylation

Abstract

Phosphorylation, a key posttranslational modification, plays a crucial role in regulating protein function and stability. However, conventional ensemble assays, such as ELISA, are unable to resolve phosphorylation at the proteoform level, thereby obscuring the heterogeneity of site-specific modifications. To address this limitation, we present a single-molecule analysis platform that combines site-specific labeling of phosphorylation sites with fluorescence resonance energy transfer (FRET) to pinpoint the location of phosphorylation sites in full-length proteins. We demonstrate the applicability of our approach on α-synuclein (αSyn), a protein implicated in neurodegenerative diseases. We can quantify the number of phosphorylation sites and identify which amino acids are phosphorylated on αSyn, depending on the type of kinase used. This single-molecule platform achieves picomolar (pM) sensitivity, enabling the detection of distinct phosphorylated proteoforms that are beyond the reach of conventional ensemble methods. Our approach enables detailed analysis of protein modifications in neurodegenerative diseases and may aid biomarker discovery.