Studies of the proteome would benefit greatly from methods to directly sequence and digitally quantify proteins and detect posttranslational modifications with single-molecule sensitivity. Here, we demonstrate single-molecule protein sequencing using a dynamic approach in which single peptides are probed in real time by a mixture of dye-labeled N-terminal amino acid recognizers and simultaneously cleaved by aminopeptidases. We annotate amino acids and identify the peptide sequence by measuring fluorescence intensity, lifetime, and binding kinetics on an integrated semiconductor chip. Our results demonstrate the kinetic principles that allow recognizers to identify multiple amino acids in an information-rich manner that enables discrimination of single amino acid substitutions and posttranslational modifications. With further development, we anticipate that this approach will offer a sensitive, scalable, and accessible platform for single-molecule proteomic studies and applications.
Comprehensive SummaryMonitoring the dynamics of a single molecule provides unique insights into the fundamental physical and chemical properties of individual molecules. Over the past few decades, various approaches have been developed to enable the real‐time and high‐resolution detection at the single‐molecule level. Among them, electrical and optical methods are the most promising tools, for the reason that electrical detection offers high resolution while optical technology provides non‐invasive, targeted measurement with the added benefit of visualization. In this review, we summarized the current state‐of‐the‐art electrical and optical techniques for single‐molecule measurement and discussed their applications in detecting dynamic events such as conformational isomerizations, intermolecular interactions, chemical reactions, and biomolecular activities. In addition, we discussed the challenges and opportunities in this area and proposed possible directions for future development.
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