Proteolysis‐Based Biomarker Repertoire of the Neurofilament Proteome

Neurodegeneration presents a significant challenge in ageing populations, often being detected too late for effective intervention. Biomarkers have shown great potential in addressing this issue, with neurofilament (Nf) proteins emerging as validated biomarkers presently transitioning from research to routine laboratory use. Whilst advances in large-scale quantitative analyses have enabled the targeted study of proteolytic Nf fragments in blood, the complete landscape of the Nf proteolytic breakdown remains unknown. This study presents a comprehensive atlas of the human Nf isoform (Z) degradome, based on the number of known cleavage sites (x). The full scale of the Nf degradome is described by the formula: Z = ((x + 1) × (x + 2)/2) - 1. The resulting neurofilament degradome atlas (NDA) was validated through a triple-layer approach using in vitro data (open access at: https://doi.org/10.5522/04/25689378.v1). The NDA offers valuable applications in biomarker detection, targeted antibody development, exploration of autoimmunity and understanding Nf aggregate formation. Analysis of the Nf degradome reveals novel insights into neurodegenerative diseases by investigating peptide pools affected by genetic mutations in the Nf genome and alterations in proteolytic pathways. The annotated NDA is publicly available as a database resource, supporting advancements in affinity-based biomarker tests through informed peptide selection and minimising biases in label-free approaches. In conclusion, this study highlights the biological significance of a dynamic pool of coexisting proteolytic Nf peptides, providing a framework that can be applied to other proteins.