Enhanced identification of Tau acetylation and phosphorylation with an engineered aerolysin nanopore

Abstract

Posttranslational modifications (PTMs) affect protein function/dysfunction, playing important roles in the occurrence and development of tauopathies including Alzheimer's disease. PTM detection is significant and still challenging due to the requirements of high sensitivity to identify the subtle structural differences between modifications. Herein, in terms of the unique geometry of the aerolysin (AeL) nanopore, we elaborately engineered a T232K AeL nanopore to detect the acetylation and phosphorylation of Tau segment (Pep). By replacing neutral threonine (T) with positively charged lysine (K) at the 232 sites, the T232K and K238 rings of this engineered T232K AeL nanopore corporately work together to enhance electrostatic trapping of the acetylated and phosphorylated Tau peptides. Translocation speed of the monophosphorylated Pep‐P was decelerated by up to 46 folds compared to the wild‐type (WT) AeL nanopore. The prolonged residences within the T232K AeL nanopore enabled to simultaneously identify the monoacetylated Pep‐Ac, monophosphorylated Pep‐P, di‐modified Pep‐P‐Ac and non‐modified Pep. The tremendous potential is demonstrated for PTM sensing by manipulating non‐covalent interactions between nanopores and single analytes.

An engineered third electrostatic constriction of aerolysin to manipulate heterogeneously charged peptide transport

“…By introducing extra positive charged lysine into the nanopore, a T232K mutant AeL achieves neutral peptide (Tau306-316) sensing. 26 Results show that the capture frequency increased compared with WT AeL. But the neutral peptide bumped out from the pore opening due to the strong repelling force, leading to a short duration below 1 ms. Herein, we design a third electrostatic constriction AeL that works together with the two pre-existing sensitive regions to control the capture and translocation of heterogeneously charged peptides.…”

Localized Nanopore Fabrication via Controlled Breakdown

“…Commonly used biological pores, such as α-hemolysin (αHL) [ 30 , 31 , 32 , 33 , 34 , 35 ], mycobacterium smegmatis porin A (MspA) [ 36 , 37 , 38 ], aeromonas hydrophila Aerolysin (AeL) [ 39 , 40 ], and bacteriophage phi29 (Phi29) [ 41 , 42 ], have relatively small constrictions with diameters smaller than 4 nm. The small constrictions of biological nanopores make them a perfect candidate for detecting small analytes such as metal ions, single polymer chains [ 35 ], DNA molecules [ 31 , 32 , 33 , 36 , 41 , 42 ], peptides [ 38 , 43 , 44 ], and unfolded proteins [ 30 ]. Moreover, these biological nanopores have a well-defined pore shape that is highly reproducible, and the proteins used to create biological nanopores can be harvested using synthetic biology techniques [ 2 , 3 , 6 ].…”

Highlights on the current state of proteomic detection and characterization with nanopore sensors

“…Huo et al [4] investigate engineered aerolysin (AeL) nanopores for the detection of post translational modifications of peptides—specifically acetylation and phosphorylation modifications to Tau proteins that have been implicated in Alzheimer's disease. This is an example of the power of nanopore sensing where distinguishing between modified peptides has proved challenging for more established techniques (i.e., mass spectrometry).…”