Selective Detection and Characterization of Small Cysteine-Containing Peptides with Cluster-Modified Nanopore Sensing

Abstract

It was recently demonstrated that one can monitor ligand-induced structure fluctuations of individual thiolate-capped gold nanoclusters using resistive-pulse nanopore sensing. The magnitude of the fluctuations scales with the size of the capping ligand, and it was later shown one can observe ligand exchange in this nanopore setup. We expand on these results by exploring the different types of current fluctuations associated with peptide ligands attaching to tiopronin-capped gold nanoclusters. We show here that the fluctuations can be used to identify the attaching peptide through either the magnitude of the peptide-induced current jumps or the onset of high-frequency current fluctuations. Importantly, the peptide attachment process requires that the peptide contains a cysteine residue. This suggests that nanopore-based monitoring of peptide attachments with thiolate-capped clusters could provide a means for selective detection of cysteine-containing peptides. Finally, we demonstrate the cluster-based protocol with various peptide mixtures to show that one can identify more than one cysteine-containing peptide in a mixture.

Cluster-Enhanced Nanopore Sensing of Ovarian Cancer Marker Peptides in Urine

“…Larger peptides (>10 residues) ejected onto the cluster typically yield a single attachment and long-lived fluctuating states. 27 Figure 2A highlights a typical current trace and corresponding all-points histogram (Figure 2B) of the filtered data (red trace, Figure 2A) from a so-called "low-frequency" peptide (P9C6; see Section 1, Table 1 in the Supporting Information). Numerous attachments of this peptide yield current steps whose magnitude are measured from the peak positions in Figure 2B.…”

“…In this paper, we begin by detecting small cancer marker peptides (<10 residues) and peptides corresponding to proteolytic cleavage products of larger biomarker peptides. These peptides give rise to step-like fluctuations with magnitudes that scale with peptide mass. , This shows promise, but the fact that a single data point results from each current step (i.e., one peptide yields one data point) limits the applicability of cluster enhanced nanopore detection. Therefore, we also report on the detection of larger cancer marker peptides (>12 residues), which give rise to so-called “high-frequency” fluctuations .…”

Engineering Biological Nanopore Approaches toward Protein Sequencing

“…This was later expanded upon to better understand what physical and chemical properties of peptides give rise to different types of fluctuations upon peptide-cluster linkage. This research found that the presence of a cysteine residue on the peptide was required for its attachment to the nanocluster and that the peptide-cluster interaction resulted in either stepwise current transitions or high-frequency one- or two-state fluctuations, depending on the given peptide (Figure b) . The nature of these current patterns was governed by various factors, such as the peptide’s mass, charge, sequence, length, and configuration.…”

“…This research found that the presence of a cysteine residue on the peptide was required for its attachment to the nanocluster and that the peptide-cluster interaction resulted in either stepwise current transitions or high-frequency one-or two-state fluctuations, depending on the given peptide (Figure 3b). 140 The nature of these current patterns was governed by various factors, such as the peptide's mass, charge, sequence, length, and configuration. Both types of peptide-induced signals could be analyzed to identify the peptides in mixtures.…”

“…The error bars in panels a and b represent standard deviations calculated from at least three independent repeats. Data in the boxes are extracted with permission from the following references: (a) (Copyright 2019 Nature Publishing Group), § (b) (Copyright 2022 American Chemical Society), (c) (Copyright 2021 Nature Publishing Group), § (d) (Copyright 2022 American Chemical Society), (e) (Copyright 2022 Springer), (f) (Copyright 2022 Royal Society of Chemistry), (g) (Copyright 2019 American Chemical Society), (h) (Copyright 2022 American Chemical Society). All corresponding schematic diagrams are created with .…”