Apparatuses and methods involving protein exploration through proteolysis and nanopore translocation
Inventors
Todd, Kathryn G. • Puster, Matthew D. • Laderoute, Keith • Luebke, Kevin • Huber, David • Yadav, Maneesh
Assignees
Publication Number
US-12163948-B2
Publication Date
2024-12-10
Expiration Date
2038-12-21
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Abstract
Embodiments in accordance with the present disclosure are directed to methods and apparatuses used for evaluating a protein in a sample using proteolysis and translocation. An example method includes isolating a single protein from a sample in a chamber having at least a first well and a second well separated from the first well by a membrane with a nanopore, the nanopore providing fluidic communication between the first and second wells. The single protein is cleaved into a plurality of peptide fragments via exposure to a protease in the first well, and translocated through the nanopore by applying an electric potential to the nanopore in the chamber after cleavage of the single protein by the protease. The method further includes detecting events indicative of the translocations of the plurality of peptide fragments through the nanopore and to the second well.
Core Innovation
The invention is directed to methods and apparatuses for evaluating proteins in a sample via proteolysis and nanopore translocation. A single protein is isolated from a sample within a chamber that contains at least a first well and a second well, separated by a membrane with a nanopore. This nanopore allows fluidic communication between the wells and is used to translocate protein fragments between them.
The single protein is cleaved into several peptide fragments by exposure to a protease in the first well. After cleavage, an electric potential is applied across the nanopore, resulting in translocation of these peptide fragments to the second well. The passage of fragments is detected through changes in ionic current, and these translocation events are used to estimate cleavage sites within the protein.
The patent addresses the challenge of exploring and identifying proteins at the single-protein level, overcoming limitations of current proteomic tools that often require larger samples or more complex reagents. By iteratively using different proteases and counting the resulting fragments, the system incrementally narrows down the identity of individual proteins in a high-throughput, label-free, and parallelizable manner.
Further, both the method and apparatus support multi-chamber, multi-well operations, allowing the evaluation of many proteins simultaneously. The process is enhanced by bioinformatics models that incorporate cleavage rules, known protein databases, and device-specific error profiles to increase identification confidence and quantify concentrations without prior knowledge of sample content.
Claims Coverage
The independent claims of this patent define two main inventive features: a method for evaluating proteins using proteolysis and nanopore translocation, and an apparatus configured to perform this process with integrated fluidics and detection systems.
Method for evaluating a single protein using proteolysis and nanopore translocation
A method involving: - Isolating a single protein from a sample in a chamber with at least a first well and a second well separated by a membrane with a nanopore providing fluidic communication. - Cleaving the single protein into a plurality of peptide fragments via exposure to a protease in the first well. - Translocating the plurality of peptide fragments through the nanopore by applying an electric potential after cleavage. - Detecting events indicative of the translocations of the plurality of peptide fragments through the nanopore to the second well. - Measuring a duration of the detected events and using the measured duration to estimate the length of the peptide fragments or to detect clogging and unclog the nanopore by reversing the bias.
Apparatus for protein analysis with fluidic channels, chambers, and integrated detection
An apparatus comprising: - Flow channels arranged to direct a sample containing a plurality of proteins. - A plurality of chambers in fluidic communication with the flow channels, each chamber having at least a first well and two secondary wells separated by membranes with a nanopore providing fluidic communication. - Fluidic access circuitry to selectively provide fluidic access to the chambers and different proteases to cleave single proteins into peptide fragments. - Electrical circuitry to drive translocations of proteins and peptide fragments across the nanopores and output electrical signals indicative of these translocations. - Processing circuitry configured to detect events indicative of the translocations, cause the electrical circuitry to reverse a bias across the nanopores, repeat translocation, and detect further events.
In summary, the patent broadly covers methods and apparatuses for isolating and identifying proteins using proteolysis-induced cleavage and nanopore-based electrical detection, integrating fluidic handling, electronic detection, and iterative analysis for protein identification and fragment characterization.
Stated Advantages
Allows identification and quantification of proteins in a sample with no prior knowledge of protein content.
Enables high-throughput, parallel, and label-free protein analysis.
Can be implemented in a portable and potentially low-cost apparatus.
Sensitive enough to work with minute sample quantities, including those from fingerprints.
Can detect proteins not accessible by DNA-based methods, including those reflecting environmental and exposure history.
Facilitates rapid identification of pathogens, diagnostic markers, and bioterrorism threats without target-specific reagents.
Provides a universal platform applicable to a wide range of proteins and sample types, including low-abundance regulatory proteins.
Supports iterative and statistical refinement of identification using a bioinformatics model that incorporates device-specific errors.
Documented Applications
Personal identification in forensic science, especially when DNA is insufficient.
Detection of bioterrorism threats and identification of emerging infectious agents, including prions and toxins.
Early stage biomarker discovery and diagnostics, including pre-symptomatic detection of infection.
Personalized therapeutics and drug development based on proteome analysis.
Inventory and quantification of proteins in cells or fluid samples for research or clinical diagnostics.
Rapid and broad-spectrum screening for viruses or bioagents.
Extension of liquid biopsy capabilities to detect proteins at lower concentrations than immunoassays.
Protein inventory in extracellular vesicles for disease monitoring, including cancer.
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