Real-Time Dynamic Single-Molecule Protein Sequencing on an Integrated Semiconductor Device

Abstract:

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.

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