Abstract
Sequence identification of peptides and proteins is central to proteomics. Protein sequencing is mainly conducted by insensitive mass spectroscopy because proteins cannot be amplified, which hampers applications such as single-cell proteomics and precision medicine. The commercial success of portable nanopore sequencers for single DNA molecules has inspired extensive research and development of single-molecule techniques for protein sequencing. Among them, three challenges remain: (1) discrimination of the 20 amino acids as building blocks of proteins; (2) unfolding proteins; and (3) controlling the motion of proteins with nonuniformly charged sequences. In this context, the emergence of label-free optical analysis techniques for single amino acids and peptides by solid-state nanopores shows promise for addressing the first challenge. In this Perspective, we first discuss the current challenges of single-molecule fluorescence detection and nanopore resistive pulse sensing in a protein sequencing. Then, label-free optical methods are described to show how they address the single-amino-acid identification within single peptides. They include localized surface plasmon resonance detection and surface-enhanced Raman spectroscopy on plasmonic nanopores. Notably, we report new data to show the ability of plasmon-enhanced Raman scattering to record and discriminate the 20 amino acids at a single-molecule level. In addition, we discuss briefly the manipulation of molecule translocation and liquid flow in plasmonic nanopores for controlling molecule movement to allow high-resolution reading of protein sequences. We envision that a combination of Raman spectroscopy with plasmonic nanopores can succeed in single-molecule protein sequencing in a label-free way.
Highlights
Primary structure identification of peptides and proteins is central to protein proteomics.[1,2] protein sequencing lags seriously behind genome sequencing
Our group reported an electro-plasmonic approach to control the residence time of biomolecules in a single hot spot by trapping a gold nanourchin (AuNU) in a plasmonic nanohole.[69,70]. Both electrokinetic forces generated by applying voltage and optical forces generated from a gradient of the plasmonic resonant electromagnetic field contribute to the trapping of AuNU inside the plasmonic nanopore
When the single-molecule DNA sequencing methods are applied to sequencing proteins, the first challenge they encountered is the detection of the 20 types of amino acids in protein sequences in contrast to the four kinds of DNA bases
Summary
Primary structure identification of peptides and proteins is central to protein proteomics.[1,2] protein sequencing lags seriously behind genome sequencing. Single-molecule analysis methods for DNA sequencing face great challenges in sequencing proteins Both fluorescence and biological nanopore sensing are difficult to discriminate the 20 amino acids as building blocks of proteins. Unlike the uniformly charged DNA, which could unidirectionally translocate in the nanopore, the amino acid residues of proteins have different charges and add complexity to the control of protein movement In this regard, label-free optical methods are being integrated with the nanopores to address these challenges, which leads to the development of various solid-state nanopores to extend their analytic functions and compatibility. We will discuss briefly the manipulation of molecule translocation and the liquid flow inside plasmonic nanopores for controlling the molecule movement and achieving high-resolution reading This Perspective will focus more on those approaches that, according to the current state of the art, are closer to the goal of protein sequencing. Besides the progress in engineering biological pores, the discrimination of 20 amino acids remains an open challenge
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