Capturing Early Aggregation Transitions of Disordered Tau by Covalent Footprinting Mass Spectrometry

Tau aggregation into β-sheet-rich fibrils is a defining pathological feature of Alzheimer's disease, yet the early conformational transitions that accompany tau aggregation, which are critical for understanding aggregation mechanisms and therapeutic intervention, remain poorly resolved due to the lack of a well-suited technique for this large intrinsically disordered protein (IDP). Here, for the first time, we captured dynamic conformational changes across full-length human tau 2N4R (441 residues) present as a heterogeneous mixture of post-translationally modified forms during a 72 h aggregation by diethyl pyrocarbonate (DEPC) covalent footprinting. Recombinant expression of fresh full-length human tau 2N4R, combined with optimized DEPC footprinting and complementary proteolytic digestion, enabled near-complete (93%) sequence coverage. Peptide-level kinetics revealed domain-specific behaviors, with the microtubule-binding domain (MTBD) exhibiting the most pronounced structural transitions. Residue-level analysis mapped these changes onto the fibril core, identifying early protection of residues from β4-β6 as an early nucleation event and progressive incorporation of adjacent MTBD strands during fibril maturation. In contrast, the N-terminal, proline-rich domain (PRD), and C-terminal region remained largely solvent-exposed throughout. Together, these results demonstrate that mass spectrometry-based covalent footprinting enables direct observation of early, dynamic conformational transitions in tau, complementing structural techniques that predominantly resolve mature aggregated states.