title: Reading the primary structure of a protein with 0.07 nm3resolution using a subnanometre-diameter pore.
abstract: The primary structure of a protein consists of a sequence of amino acids and is a key factor in determining how a protein folds and functions. However, conventional methods for sequencing proteins, such as mass spectrometry and Edman degradation, suffer from short reads and lack sensitivity, so alternative approaches are sought. Here, we show that a subnanometre-diameter pore, sputtered through a thin silicon nitride membrane, can be used to detect the primary structure of a denatured protein molecule. When a denatured protein immersed in electrolyte is driven through the pore by an electric field, measurements of a blockade in the current reveal nearly regular fluctuations, the number of which coincides with the number of residues in the protein. Furthermore, the amplitudes of the fluctuations are highly correlated with the volumes that are occluded by quadromers (four residues) in the primary structure. Each fluctuation, therefore, represents a read of a quadromer. Scrutiny of the fluctuations reveals that the subnanometre pore is sensitive enough to read the occluded volume that is related to post-translational modifications of a single residue, measuring volume differences of ∼0.07 nm3, but it is not sensitive enough to discriminate between the volumes of all twenty amino acids.
authors.affiliation: Electrical Engineering, University of Notre Dame, Notre Dame, Indiana 46556, USA.
authors.forename: Eamonn
authors.initials: E
authors.lastname: Kennedy
authors.affiliation: Electrical Engineering, University of Notre Dame, Notre Dame, Indiana 46556, USA.
authors.forename: Zhuxin
authors.initials: Z
authors.lastname: Dong
authors.affiliation: Chemical Engineering, University of Notre Dame, Notre Dame, Indiana 46556, USA.
authors.forename: Clare
authors.initials: C
authors.lastname: Tennant
authors.affiliation: Electrical Engineering and Biological Science, University of Notre Dame, Notre Dame, Indiana 46556, USA.
authors.forename: Gregory
authors.initials: G
authors.lastname: Timp
mesh_terms: D000595:Amino Acid Sequence; D007091:Image Processing, Computer-Assisted; D056656:Lab-On-A-Chip Devices; D008239:Lysine / Q000737:chemistry; D008567:Membranes, Artificial; D008623:Mercaptoethanol / Q000737:chemistry; D017348:Microscopy, Electron, Scanning Transmission / Q000295:instrumentation; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods*; D011489:Protein Denaturation; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D017655:Silicon Compounds; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry
chemical_list: D008567:Membranes, Artificial; D011506:Proteins; D017655:Silicon Compounds; D012967:Sodium Dodecyl Sulfate; D008623:Mercaptoethanol; D008239:Lysine; C032734:silicon nitride