Machine Learning Assisted Simultaneous Structural Profiling of Differently Charged Proteins in a Mycobacterium smegmatis Porin A (MspA) Electroosmotic Trap

Abstract

The nanopore is emerging as a means of singlemolecule protein sensing. However, proteins demonstrate different charge properties, which complicates the design of a sensor that can achieve simultaneous sensing of differently charged proteins. In this work, we introduce an asymmetric electrolyte buffer combined with the Mycobacterium smegmatis porin A (MspA) nanopore to form an electroosmotic flow (EOF) trap. Apo-and holo-myoglobin, which differ in only a single heme, can be fully distinguished by this method. Direct discrimination of lysozyme, apo/holo-myoglobin, and the ACTR/NCBD protein complex, which are basic, neutral, and acidic proteins, respectively, was simultaneously achieved by the MspA EOF trap. To automate event classification, multiple event features were extracted to build a machine learning model, with which a 99.9% accuracy is achieved. The demonstrated method was also applied to identify single molecules of α-lactalbumin and βlactoglobulin directly from whey protein powder. This protein-sensing strategy is useful in direct recognition of a protein from a mixture, suggesting its prospective use in rapid and sensitive detection of biomarkers or real-time protein structural analysis.

Biological nanopores for single-molecule sensing

“…In biological nanopores a sufficient EOF is mostly generated through engineering proper electrostatics into the pore lumen or simply by conducting the experiment at a low or high pH ( Asandei et al., 2016 ; Huang et al., 2019 ; Wang et al., 2017b ) at which the overall charge of the pore will be more positive or negative, respectively. The EOF can in some cases be large enough to capture analytes against the electrophoretic force ( Asandei et al., 2016 ) and be used to capture peptides and proteins in α-HL ( Asandei et al., 2016 ), FraC ( Huang et al., 2019 ), MpsA ( Liu et al., 2022 ), and PlyAB ( Huang et al., 2020 ). In addition, the introduction of pore mutations contrasting the electrostatic signature of the analyte is also a solution to increase the capture rate ( Maglia et al., 2008 ).…”

Nanopore Identification of Alditol Epimers and Their Application in Rapid Analysis of Alditol-Containing Drinks and Healthcare Products

“…The MspA nanopore is conically shaped and has demonstrated superior resolution in the discrimination of epigenetic modifications, DNA lesions, , RNA structures, and protein structures. , Engineered MspA has also directly observed the coordination chemistry of a single metal ion at high resolution. ,− However, the octameric symmetry of MspA has posed a technical challenge to the introduction of a sole reactive site for sensing. In this paper, a hetero-octameric MspA nanopore containing a single phenylboronic acid (MspA-PBA) was designed, prepared, and used as an alditol sensor.…”

Application of Nanopore Sensors for Biomolecular Interactions and Drug Discovery

“…Recently, Liu et al. demonstrated EOF‐based folded protein trapping with a mutant MspA nanopore using an asymmetric electrolyte buffer setup [35] . The calcium chloride (CaCl 2 ) can accumulate on the negatively charged pore surface, and changes in the nanopore surface charge can generate EOF.…”

“…Profiling of four proteins (lysozyme, holo‐MB, ACTR/NCBD, and apo‐MB) by using nanopore events in combination with machine learning. Reprinted with permission [35] . Copyright 2022 American Chemical Society.…”