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.

Single-Molecule Peptide Identification Using Fluorescence Blinking Fingerprints

“…Similar to other recent studies in this field, 10,14 so far, we have relied on synthetic peptides that are functionalized for click chemistry. To be able to analyze naturally occurring peptides and proteins, strategies for surface immobilization and amino-acid-specific labeling have to be developed.…”

“…Nanopore-based sequencing struggles with amino acid detection accuracy, linearization of large peptides and proteins, translocation of positively charged peptides, and throughput. Single-molecule fluorescence-based techniques are massively parallelizable, but some of these methods rely on multiple cycles of chemical or enzymatic degradation, , making data acquisition long and prone to errors. Other fluorescence-based methods can detect accurately the position of selected amino acids, , but it is unclear how sensitive these methods are to the nature of PTMs present in other amino acids in the peptide.…”

Digital detection of proteins

“…Given the lack of methods for the direct molecular amplification of proteins, the counting of single protein molecules seems to be the front-runner in the race. Approaches to next-generation protein sequencing 170 and CRISPR-based detection of proteins 171 may provide alternative approaches to highly sensitive protein detection, although these methods may also benefit from single molecule detection.…”

Seven technologies to watch in 2023

“…"We could label the proteins with different fluorescent dyes and then watch molecule by molecule as we cut them away," Marcotte explains. Last year, researchers at QuantumSi, a biotechnology firm in Guilford, Connecticut, described an alternative to fluorosequencing that uses fluorescently labelled 'binder' proteins to recognize specific sequences of amino acids (or polypeptides) at the ends of proteins 2 .…”