Protein Sequencing, One Molecule at a Time

Despite tremendous gains over the past decade, methods for characterizing proteins have generally lagged behind those for nucleic acids, which are characterized by extremely high sensitivity, dynamic range, and throughput. However, the ability to directly characterize proteins at nucleic acid levels would address critical biological challenges such as more sensitive medical diagnostics, deeper protein quantification, large-scale measurement, and discovery of alternate protein isoforms and modifications and would open new paths to single-cell proteomics. In response to this need, there has been a push to radically improve protein sequencing technologies by taking inspiration from high-throughput nucleic acid sequencing, with a particular focus on developing practical methods for single-molecule protein sequencing (SMPS). SMPS technologies fall generally into three categories: sequencing by degradation (e.g., mass spectrometry or fluorosequencing), sequencing by transit (e.g., nanopores or quantum tunneling), and sequencing by affinity (as in DNA hybridization-based approaches). We describe these diverse approaches, which range from those that are already experimentally well-supported to the merely speculative, in this nascent field striving to reformulate proteomics.