Protein identification by nanopore peptide profiling

Lucas, Florian Leonardus Rudolfus; Versloot, Roderick Corstiaan Abraham; Yakovlieva, Liubov; Walvoort, Marthe T. C.; Maglia, Giovanni

doi:10.1038/s41467-021-26046-9

Cited by 78 publications

(87 citation statements)

References 53 publications

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“… 18 Single proteins are then addressed by directly reading the unfolded protein as it translocated across the nanopore or by chopping the protein into peptides and reading the fragments in sequence. 19 We also showed that the latter “chop and drop” method could potentially discriminate among the majority of proteins of the human proteome, provided that most peptides were accurately detected by the nanopore. 20 α-Helical fragaceatoxin C (FraC) nanopores were shown to efficiently capture peptides in low pH conditions regardless of their charge.…”

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confidence: 73%

“… 20 α-Helical fragaceatoxin C (FraC) nanopores were shown to efficiently capture peptides in low pH conditions regardless of their charge. 19 , 21 − 23 In turn, this allowed several proteins to be recognized by measuring the peptide spectrum originating from trypsinated proteins. Although this approach is capable of distinguishing among proteins at the single-molecule level, it is unclear whether a cylindrical β-barrel nanopore, such as those incorporated into the nanopore–unfoldase complex, can be engineered to identify proteinogenic peptides in a fashion similar to conical α-helical FraC nanopores.…”

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confidence: 99%

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β-Barrel Nanopores with an Acidic–Aromatic Sensing Region Identify Proteinogenic Peptides at Low pH

et al. 2022

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Biological nanopores are emerging as sensitive single-molecule sensors for proteins and peptides. The heterogeneous charge of a polypeptide chain, however, can complicate or prevent the capture and translocation of peptides and unfolded proteins across nanopores. Here, we show that two β-barrel nanopores, aerolysin and cytotoxin K, cannot efficiently detect proteinogenic peptides from a trypsinated protein under a wide range of conditions. However, the introduction of an acidic–aromatic sensing region in the β-barrel dramatically increased the dwell time and the discrimination of peptides in the nanopore at acidic pH. Surprisingly, despite the fact that the two β-barrel nanopores have a similar diameter and an acidic–aromatic construction, their capture mechanisms differ. The electro-osmotic flow played a dominant role for aerolysin, while the electrophoretic force dominated for cytotoxin K. Nonetheless, both β-barrel nanopores allowed the detection of mixtures of trypsinated peptides, with aerolysin nanopores showing a better resolution for larger peptides and cytotoxin K showing a better resolution for shorter peptides. Therefore, this work provides a generic strategy for modifying nanopores for peptide detection that will be most likely be applicable to other nanopore-forming toxins.

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β-Barrel Nanopores with an Acidic–Aromatic Sensing Region Identify Proteinogenic Peptides at Low pH

et al. 2022

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“…One method is to adopt a protein fingerprinting approach like the shotgun proteomics used in mass spectrometry. 8 The other strategy employs DNA motor enzymes to ratchet a peptide–oligonucleotide conjugate into the nanopore. 6,7,9 The former method requires the digestion of the protein.…”

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confidence: 99%

An engineered third electrostatic constriction of aerolysin to manipulate heterogeneously charged peptide transport

2022

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“…Ion currents have been widely used to monitor various biosamples, such as cells [13], bacteria, viruses, peptides [14], and DNAs [15,16] passing through nano/micropores. In addition to nanopores based on biomaterials [17], solid-state nanopores have also been employed [18].…”

Section: Introductionmentioning

confidence: 99%

Ion Conductance-Based Perfusability Assay of Vascular Vessel Models in Microfluidic Devices

et al. 2021

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We present a novel methodology based on ion conductance to evaluate the perfusability of vascular vessels in microfluidic devices without microscopic imaging. The devices consisted of five channels, with the center channel filled with fibrin/collagen gel containing human umbilical vein endothelial cells (HUVECs). Fibroblasts were cultured in the other channels to improve the vascular network formation. To form vessel structures bridging the center channel, HUVEC monolayers were prepared on both side walls of the gel. During the culture, the HUVECs migrated from the monolayer and connected to the HUVECs in the gel, and vascular vessels formed, resulting in successful perfusion between the channels after culturing for 3–5 d. To evaluate perfusion without microscopic imaging, Ag/AgCl wires were inserted into the channels, and ion currents were obtained to measure the ion conductance between the channels separated by the HUVEC monolayers. As the HUVEC monolayers blocked the ion current flow, the ion currents were low before vessel formation. In contrast, ion currents increased after vessel formation because of creation of ion current paths. Thus, the observed ion currents were correlated with the perfusability of the vessels, indicating that they can be used as indicators of perfusion during vessel formation in microfluidic devices. The developed methodology will be used for drug screening using organs-on-a-chip containing vascular vessels.

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Protein identification by nanopore peptide profiling

Cited by 78 publications

References 53 publications

β-Barrel Nanopores with an Acidic–Aromatic Sensing Region Identify Proteinogenic Peptides at Low pH

β-Barrel Nanopores with an Acidic–Aromatic Sensing Region Identify Proteinogenic Peptides at Low pH

An engineered third electrostatic constriction of aerolysin to manipulate heterogeneously charged peptide transport

Ion Conductance-Based Perfusability Assay of Vascular Vessel Models in Microfluidic Devices

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