Presently,proteins are identified by cleaving them with proteases,measuring the mass to charge ratio of the fragments with a mass spectrometer,and matching the fragments to segments within known proteins in databases....Presently,proteins are identified by cleaving them with proteases,measuring the mass to charge ratio of the fragments with a mass spectrometer,and matching the fragments to segments within known proteins in databases.We earlier demonstrated that a nanometer-scale pore formed by aerolysin(AeL)can discriminate between,and therefore identify,three similar size proteins from their trypsin-cleaved polypeptide fragments.With this nanopore-protease method,the protein’s identity is instead determined from characteristic ionic current blockade patterns caused by the polypeptide fragments that enter the nanopore.The results also suggested that not all of the theoretically expected cleavage products partition into the pore.To better understand the mechanism by which polypeptide fragments are captured,and how different polypeptides reduce the pore’s ionic current,we studied the effects of 11 identical length polypeptides with different net charges and charge distributions.We show that under certain experimental conditions,negative,positive,and neutral polypeptides are driven into the AeL pore by the same applied voltage polarity.The capture rate and dwell time of polypeptides in the pore depend strongly on the ionic strength,the magnitude of the applied voltage,and the net charge and charge distribution of the polypeptides.The dwell time distribution depends nonmonotonically on the applied voltage(regardless of the polymer’s net charge),and its maximum value depends on the polypeptide net charge and charge distribution.The maximum dwell time for different polypeptides does not occur at the same applied voltage amplitude,which conceivably might complicate the detection and discrimination of some polypeptide fragments.Although additional experiments,computer simulations,and artificial intelligence research are needed to better understand how to optimize the partitioning of enzymatically cleaved fragments into the AeL nanopore,the method is still capable of accurately identifying proteins.展开更多
基金supported by the Agence Nationale de la Recherche ANR(ANR-17-CE09-0032-01 to A.O.,M.P.-G.,and F.P.)NIST Office of Law Enforcement Standards,and a Marie Skłodowska-Curie/Freiburg Institute for Advanced Studies Senior Fellowship(both to J.J.K.)We thank F.Gisou van der Goot(Ecole Polytechnique Federale de Lausanne,Switzerland)for providing the pET22b-proAL plasmid containing the proaerolysin sequence.
文摘Presently,proteins are identified by cleaving them with proteases,measuring the mass to charge ratio of the fragments with a mass spectrometer,and matching the fragments to segments within known proteins in databases.We earlier demonstrated that a nanometer-scale pore formed by aerolysin(AeL)can discriminate between,and therefore identify,three similar size proteins from their trypsin-cleaved polypeptide fragments.With this nanopore-protease method,the protein’s identity is instead determined from characteristic ionic current blockade patterns caused by the polypeptide fragments that enter the nanopore.The results also suggested that not all of the theoretically expected cleavage products partition into the pore.To better understand the mechanism by which polypeptide fragments are captured,and how different polypeptides reduce the pore’s ionic current,we studied the effects of 11 identical length polypeptides with different net charges and charge distributions.We show that under certain experimental conditions,negative,positive,and neutral polypeptides are driven into the AeL pore by the same applied voltage polarity.The capture rate and dwell time of polypeptides in the pore depend strongly on the ionic strength,the magnitude of the applied voltage,and the net charge and charge distribution of the polypeptides.The dwell time distribution depends nonmonotonically on the applied voltage(regardless of the polymer’s net charge),and its maximum value depends on the polypeptide net charge and charge distribution.The maximum dwell time for different polypeptides does not occur at the same applied voltage amplitude,which conceivably might complicate the detection and discrimination of some polypeptide fragments.Although additional experiments,computer simulations,and artificial intelligence research are needed to better understand how to optimize the partitioning of enzymatically cleaved fragments into the AeL nanopore,the method is still capable of accurately identifying proteins.
