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Abstract

Protein-based therapeutics, such as antibodies and nanobodies, are not encoded in reference genomes, challenging their accurate characterization via standard proteomics. Current methods rely on indirect inference, fragmented outputs, and labor-intensive workflows, which hinder functional insights and routine application. Here, we present a generalizable, end-to-end workflow for direct protein sequencing, combining streamlined sample preparation, AI-driven de novo peptide sequencing, and tailored assembly to reconstruct contiguous protein sequences. A novel composite scoring framework prioritises longer assemblies and coverage, enhancing accuracy and reducing ambiguity. Validation across diverse protein modalities demonstrates its utility and ability to robustly sequence functionally critical regions of selected proteins. This workflow represents an advance in precision proteomics with promising applications in therapeutic discovery, immune profiling, and protein science.

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