Multiple rereads of single proteins at single–amino acid resolution using nanopores

Abstract

Reading amino acids by nanopore Nanopore technology enables sensing of minute chemical changes at the single-molecule level by detecting differences in an ion current as molecules are drawn through a membrane-embedded pore. The sensitivity is sufficient to discriminate between nucleotide bases in nanopore sequencing, and other applications of this technology are promising. Brinkerhoff et al . developed a nanopore-based, single-molecule approach in which a protein was sequentially scanned in single-amino-acid steps through the narrow construction of a nanopore, and ion currents were monitored to resolve differences in the amino acid sequence along the peptide backbone (see the Perspective by Bošković and Keyser). The peptide reader was capable of reliably detecting single-amino-acid substitutions within individual peptides. An individual protein could be re-read many times, yielding very high read accuracy in variant identification. These proof-of-concept nanopore experiments constitute a promising basis for the development of a single-molecule protein sequencer. —DJ

Planar Boronic Graphene and Nitrogenized Graphene Heterostructure for Protein Stretch and Confinement

“…Following the similar approach in our previous studies [ 17 , 24 , 30 , 31 , 32 ], we performed a pre-equilibrium simulation of the conformation of each IDP fragment from solution to adsorption on a BC 3 nanosheet, and then extended the BC 3 nanosheet (along with the peptide) to construct the BC 3 /C 3 N/BC 3 planar heterostructure. After that, the entire system is placed in a box with a size of 16.2 × 14.1 × 5.0 nm 3 [ 3 ] and solvated with 100 mM KCl electrolyte, which contains approximately 66,000 atoms. In addition, to explore the difference in hydrophilic or hydrophobic properties between BC 3 and C 3 N, we constructed two systems of BC 3 or C 3 N in a water box with a size of 4.9 × 4.2 × 5.0 nm 3 [ 3 ].…”

“…After that, the entire system is placed in a box with a size of 16.2 × 14.1 × 5.0 nm 3 [ 3 ] and solvated with 100 mM KCl electrolyte, which contains approximately 66,000 atoms. In addition, to explore the difference in hydrophilic or hydrophobic properties between BC 3 and C 3 N, we constructed two systems of BC 3 or C 3 N in a water box with a size of 4.9 × 4.2 × 5.0 nm 3 [ 3 ]. Later, to characterize the interface behavior of water molecules on the planar heterostructure, we additionally constructed a heterostructure-water system with a box size of 16.2 × 14.1 × 5.0 nm 3 [ 3 ].…”

“…Protein sequencing at the single-molecule level is crucial for personalized medicines and the detection of post-translational modifications in proteins [ 1 , 2 , 3 , 4 ]. Recently, several single-molecule techniques such as atomic force microscopy (AFM) [ 5 , 6 ], quantum tunneling [ 7 , 8 , 9 ], and nanopore [ 3 , 10 , 11 ] have been proposed for protein sequencing, which allow the direct read-out of structural differences of individual amino acids.…”

“…Protein sequencing at the single-molecule level is crucial for personalized medicines and the detection of post-translational modifications in proteins [ 1 , 2 , 3 , 4 ]. Recently, several single-molecule techniques such as atomic force microscopy (AFM) [ 5 , 6 ], quantum tunneling [ 7 , 8 , 9 ], and nanopore [ 3 , 10 , 11 ] have been proposed for protein sequencing, which allow the direct read-out of structural differences of individual amino acids. Although promised to be with high accuracy and low cost, a large gap still resides between these proof-of-principle methods and the ultimate sensitivity for the discrimination of 20 different amino acids.…”

Resolving isomeric posttranslational modifications using a nanopore

“…Harnessing of nanopore technology for applications in proteomics has been an active area of research for some time 2,25 , including the detection of PTMs [26][27][28][29] . Sequencing proteins by nanopore threading, which would in principle allow detection of PTMs in a site-specific manner, is presently in early stages of development, but still faces the lack of processive motors suitable for moving a polypeptide chain through the nanopore and of physical mechanisms for keeping an unevenly charged peptide taut through the nanopore's constriction 3,4,25,30 . Here we show that site-specific detection of histone PTMs is feasible by whole-molecule nanopore sensing of peptide fragments, requiring neither sequential threading nor uniform charge of the analyte.…”