Large-scale single-molecule analysis of tau proteoforms.

Area of Science:

• Biochemistry and Molecular Biology
• Neuroscience
• Proteomics

Background:

• Proteins exist as diverse proteoforms due to genetic variation, alternative splicing, and post-translational modifications.
• Current single-molecule protein analysis methods lack the resolution to capture this complexity.
• Understanding tau proteoform diversity is crucial for neurodegenerative disease research.

Purpose of the Study:

• To introduce Iterative Mapping of Proteoforms (IMaP), a novel method for high-throughput single-molecule proteoform analysis.
• To characterize tau proteoform group profiles, including phosphorylation patterns, in neuronal and disease samples.
• To provide insights into protein regulation and disease mechanisms at the single-molecule level.

Main Methods:

• Developed Iterative Mapping of Proteoforms (IMaP) for massively-parallel interrogation of single-protein molecules.
• Utilized iterative probing with 12 site-specific, fluorescently labeled antibodies against tau protein variants and phosphorylation sites.
• Applied IMaP to neuronal models, human samples, and Alzheimer's disease patient samples.

Main Results:

• IMaP can measure 4,096 potential proteoform groups with high sensitivity (0.1% abundance), reproducibility (CV <5.5%), and dynamic range (>3 orders of magnitude).
• Identified 130 distinct tau proteoform groups, including up to six phosphorylation events, in biological samples.
• Observed non-random, site-specific phosphorylation patterns and preferential co-occurrence of certain phosphorylation events (e.g., pT217 with pT181).
• Detected a specific multi-phosphorylation pattern in Alzheimer's disease samples suggesting a sequential pathological tau modification pathway.

Conclusions:

• Iterative Mapping of Proteoforms (IMaP) enables unprecedented resolution of single-molecule proteoform complexity.
• The findings reveal ordered phosphorylation processes in tau protein, relevant to neurodegenerative disease pathogenesis.
• IMaP has significant implications for understanding protein regulation and developing diagnostics for neurodegenerative diseases.