Proteomics has emerged as a crucial field for understanding diseases, developing diagnostics, and designing effective therapeutics. Rapid, agnostic detection of future unknown protein-based biological threats necessitates the development of single-molecule protein sequencing technology that can differentiate the 20 canonical amino acids and beyond. While DNA sequencing has become widely accessible, protein sequencing has lagged behind due to significant technical complexity, including the 20 primary amino acids, numerous non-canonical and modified amino acids, hundreds of post-translational modifications (PTMs), the inability to amplify samples, a high dynamic range in biological samples, and variable solubility (1). The Defense Advanced Research Project Agency (DARPA) seeks to build a technology that can directly read individual biological polymers (e.g., proteins) through the use of nanopore-based platforms. The technology will enable the identification of unknown biomolecules in real time with reliable devices to address detection gaps for protein-based threats. The current State of the Art (SOA) in proteomic polymer sequencing is defined by mass spectrometry (MS)-based platforms that allow deep qualitative and quantitative analysis of protein sequence, expression, interactions and post-translational modifications. The predominant “bottom-up” strategy involves enzymatic digestion of proteins into peptides, which are subsequently separated by liquid chromatography prior to identification and quantification using tandem MS. Despite demonstrating great utility, this destructive process often provides incomplete sequence coverage and critical information regarding full-length protein isoforms and the combinatorial arrangement of post-translational modifications on a single protein molecule is lost. Recent advances in data-independent acquisition approaches, run on SOA instrumentation like Orbitraps and TimsTOFs, now enable the quantification of over 10,000 proteins from bulk samples and can identify over 5,000 proteins at the single-cell level (2). Despite this remarkable capability, the technology is constrained by limited dynamic range making the detection of low-abundance proteins challenging. Any novel, single-molecule protein sequencing approach will ultimately be measured against these established technological benchmarks that depend upon inferring protein identities from peptide fragments. Novel nanopore-based technologies offer a promising path towards direct, real-time, single-molecule sequencing, potentially overcoming many of the limitations of current MS-based methods (3,4,5). To meet the need for rapid detection of unknown biological threats, DARPA seeks biological nanopore-based technology to advance our capability to read proteins, including novel sample preparation technologies, engineered protein-based motors and nanopores for molecular transit, and machine learning models for deconvoluting electronic signals.
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