Nanopore‐Based High Resolution Detection of Multiple Post‐Translational Modifications in Proteins
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Protein post‐translational modifications (PTMs) play crucial roles in various cellular processes. Despite their significance, only a few PTMs have been extensively studied at the proteome level, primarily due to the scarcity of reliable, convenient, and low‐cost sensing methods. Here, we present a straightforward and effective strategy for detecting PTMs on short peptides through host‐guest interaction‐assisted nanopore sensing. Our results demonstrate that the identity of 13 types of PTMs in a specific position of a phenylalanine‐containing peptide could be determined via current blockage during translocation of the peptide through α‐hemolysin nanopores in the presence of cucurbit[7]uril. Furthermore, we extend this strategy by incorporating a short peptide into the probe, enabling the discrimination of various PTMs, positional isomers, and even multiple PTMs on the target peptide. With ongoing improvements, our method holds promise for practical applications in sensing PTMs in biologically relevant samples, offering an efficient alternative to traditional mass spectrometry approaches.Keywords:
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Proteome
Posttranslational modification
Abstract Nanopore sensing is an attractive, label‐free approach that can measure single molecules. Although initially proposed for rapid and low‐cost DNA sequencing, nanopore sensors have been successfully employed in the detection of a wide variety of substrates. Early successes were mostly achieved based on two main strategies by 1) creating sensing elements inside the nanopore through protein mutation and chemical modification or 2) using molecular adapters to enhance analyte recognition. Over the past five years, DNA molecules started to be used as probes for sensing rather than substrates for sequencing. In this Minireview, we highlight the recent research efforts of nanopore sensing based on DNA‐mediated characteristic current events. As nanopore sensing is becoming increasingly important in biochemical and biophysical studies, DNA‐based sensing may find wider applications in investigating DNA‐involving biological processes.
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Inside Back Cover In article number 2100542, Tsutsui, Baba, Kawai, and co-workers demonstrated the use of a 3D integrated nanopore system for in situ detections of single molecule proteins and DNA in a cell by ionic current. With the proven potential of nanopore sequencing, the present device technology promises amplification free genome analysis that may revolutionize biology and medicine through uncovering genetic variations at a single cell level.
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The α-hemolysin nanopore is a promising sensor for ultra-rapid sequencing of DNA strands within nanopores. By using immobilized synthetic oligonucleotides, it is shown that additional sequence information can be gained when two recognition sites, rather than one, are employed within a single nanopore (see picture).
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Nanopore single molecule sequencing(SMS) is considered as one of the most promising third generation DNA sequencing method by virtue of fast sequencing and low cost. As the most significant part of the sequencing system, the synthetic solid nanopore has recently been the research highlight in the field of nanopore DNA sequencing because of its fine-controlled size, high reliability and wide applicability. Also, its fabrication method is currently one of the main challenges in this field. In this article, the principles of the nanopore SMS are introduced followed by the review of the difficulties and challenges in the current research stage with emphasis on the nanopore fabrication methods.
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DNA sequencing; Single molecule sequencing; Nanopore; Fabrication
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Rapid progresses have been achieved in one of the most promising third-generation sequencing methods: nanopore sequencing during recent years. Optical nanopore sequencing shows visible potential as the latest method. By optically encoding the ionic flux through protein nanopores, such as α-haemolysin and MspA, in a single droplet interface bilayer, the discrimination and detection of nucleic acid sequences can be parallelized. Nanopore blockades can discriminate between DNAs with sub-picoampere equivalent resolution, and specific miRNA sequences can be identified by differences in unzipping kinetics. If completely developed, this method will greatly increase the speed of sequencing after overcoming the sacrifices in device size and cost.
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In recent years, nanopore technology has become increasingly important in the field of life science and biomedical research. By embedding a nano-scale hole in a thin membrane and measuring the electrochemical signal, nanopore technology can be used to investigate the nucleic acids and other biomacromolecules. One of the most successful applications of nanopore technology, the Oxford Nanopore Technology, marks the beginning of the fourth generation of gene sequencing technology. In this review, the operational principle and the technology for signal processing of the nanopore gene sequencing are documented. Moreover, this review focuses on the applications using nanopore gene sequencing technology, including the diagnosis of cancer, detection of viruses and other microbes, and the assembly of genomes. These applications show that nanopore technology is promising in the field of biological and biomedical sensing.
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Much tremendous break through have been obtained in recent years for nanopore sequencing to achieve the goal of $1000 genome. As a method of single molecule sequencing, nanopore sequencing can discriminate the individual molecules of the target DNA strand rapidly due to the current blockages by translocating the nucleotides through a nano-scale pore. Both the protein-pores and solid-state nanopore channels which called single nanopore sequencing have been studied widely for the application of nanopore sequencing technology. This review will give a detail representation to protein nanopore and solid-state nanopore sequencing. For protein nanopore sequencing technology, we will introduce different nanopore types, device assembly and some challenges still exist at present. We will focus on more research fields for solid-state nanopore sequencing in terms of materials, device assembly, fabricated methods, translocation process and some specific challenges. The review also covers some of the technical advances in the union nanopore sequencing, which include nanopore sequencing combine with exonuclease, hybridization, synthesis and design polymer.
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