Resolving Isomeric Posttranslational Modifications Using a Biological Nanopore as a Sensor of Molecular Shape

Ensslen, Tobias; Sarthak, Kumar; Aksimentiev, Aleksei; Behrends, Jan C.

doi:10.1021/jacs.2c06211

Cited by 38 publications

(34 citation statements)

References 50 publications

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“…As a known family of biomarkers with a phosphorylated form that has physiological relevance, the fibrinopeptide A family serves as a medically important challenge for the proof-of-concept for this approach. We use a powerful sensor for biomolecule characterization of the aerolysin nanopore , that was previously used for the discrimination of biomolecule size and sequence, ,, chemical modifications, ,,− and conformation. ,− …”

Section: Introductionmentioning

confidence: 99%

Nanopore Discrimination of Coagulation Biomarker Derivatives and Characterization of a Post-Translational Modification

Stierlen

Greive²,

Bacri

et al. 2023

ACS Cent. Sci.

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One of the most important health challenges is the early and ongoing detection of disease for prevention, as well as personalized treatment management. Development of new sensitive analytical point-of-care tests are, therefore, necessary for direct biomarker detection from biofluids as critical tools to address the healthcare needs of an aging global population. Coagulation disorders associated with stroke, heart attack, or cancer are defined by an increased level of the fibrinopeptide A (FPA) biomarker, among others. This biomarker exists in more than one form: it can be post-translationally modified with a phosphate and also cleaved to form shorter peptides. Current assays are long and have difficulties in discriminating between these derivatives; hence, this is an underutilized biomarker for routine clinical practice. We use nanopore sensing to identify FPA, the phosphorylated FPA, and two derivatives. Each of these peptides is characterized by unique electrical signals for both dwell time and blockade level. We also show that the phosphorylated form of FPA can adopt two different conformations, each of which have different values for each electrical parameter. We were able to use these parameters to discriminate these peptides from a mix, thereby opening the way for the potential development of new point-of-care tests.

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Section: Introductionmentioning

confidence: 99%

Nanopore Discrimination of Coagulation Biomarker Derivatives and Characterization of a Post-Translational Modification

Stierlen

Greive²,

Bacri

et al. 2023

ACS Cent. Sci.

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“…Some of them were also applied to study peptide/protein post-translational modifications including phosphorylation, [11] glycosylation, [12] acetylation, [13] and methylation. [14] Recently, the aerolysin nanopore successfully identified the variations of single standard amino acids, which were purposefully positioned at the C-terminus of a polyarginine carrier. [15] By employing a DNA helicase or polymerase motor to control the translocations of the specially designed DNA-peptide conjugates, several proof-of-concept studies demonstrated the capability of Mycobacterium smegmatis porin A (MspA) nanopores in single-molecule peptide and protein sequencing.…”

Section: Introductionmentioning

confidence: 99%

3D Blockage Mapping for Identifying Familial Point Mutations in Single Amyloid‐β Peptides with a Nanopore

Xin

Liu

et al. 2022

Angewandte Chemie

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Accurate discrimination of amyloid-β (Aβ) peptides containing familial point mutations would advance the knowledge of their roles in early-onset Alzheimer's disease. Herein, we simultaneously identified the mutant A21G, E22G, E22Q, and the wild-type (WT) Aβ 18-26 peptides with aerolysin nanopore using a 3D blockage mapping strategy. The standard deviation of current blockade fluctuations (σ b ) was proposed as a new supplement to current blockage (I b /I 0 ) and duration time (t D ) to profile the blockage characteristics of single molecules. Although the WT and A21G Aβ 18-26 are indistinguishable in a traditional I b /I 0 -t D 2D description, ~87 % of the blockade events can be accurately classified with half reduction of false identification using a combination of I b /I 0 , t D, and σ b . This work offers an easy and reliable strategy to promote nanopore sensitivity of peptide mutants, leading to a more precise analysis of pathogenic mutations for developing effective diagnosis and treatment.

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“…Considering that cation−π and π-stacking interactions are expected to increase the dwell time of glycopeptides in the nanopore, a Frac nanopore combining an aromatic sensing region was developed for the quantification of protein glycosylation at high electrolyte concentrations. A recent research study about PTM of human histone H4 protein using aerolysin pore has been reported based on the principle of molecular shape sensing.…”

mentioning

confidence: 99%

“…Considering that cation−π and π-stacking interactions are expected to increase the dwell time of glycopeptides in the nanopore, a Frac nanopore 31 combining an aromatic sensing region was developed for the quantification of protein glycosylation at high electrolyte concentrations. A recent research study 32 about PTM of human histone H4 protein using aerolysin pore has been reported based on the principle of molecular shape sensing. However, the post-translational modified groups mentioned above are either negatively charged or bulky, the inherent characterization 9 of the methylation of arginine such as not changing the charge of the peptide chain and the small size and molecular weight of a methyl group makes it difficult to identify.…”

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confidence: 99%

Recognition Receptor for Methylated Arginine at the Single Molecular Level

et al. 2023

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Among the various types of post-translational modifications (PTMs), methylation is the simple functionalized one that regulates the functions of proteins and affects interactions of protein–protein and protein–DNA/RNA, which will further influence diverse cellular processes. The methylation modification has only a slight effect on the size and hydrophobicity of proteins or peptides, and it cannot change their net charges at all, so the methods for recognizing methylated protein are still limited. Here, we designed a recognition receptor consisting of a α-hemolysin (α-HL) nanopore and polyamine decorated γ-cyclodextrin (am8γ-CD) to differentiate the methylation of peptide derived from a heterogeneous nuclear ribonucleoprotein at the single molecule level. The results indicate that the modification of a methyl group enhances the interaction between the peptide and the recognition receptor. The results of molecular simulations were consistent with the experiments; the methylated peptide interacts with the receptor strongly due to the more formation of hydrogen bonds. This proposed strategy also can be used to detect PTM in real biological samples and possesses the advantages of low-cost and high sensitivity and is label-free. Furthermore, the success in the construction of this recognition receptor will greatly facilitate the investigation of pathogenesis related to methylated arginine.

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Resolving Isomeric Posttranslational Modifications Using a Biological Nanopore as a Sensor of Molecular Shape

Cited by 38 publications

References 50 publications

Nanopore Discrimination of Coagulation Biomarker Derivatives and Characterization of a Post-Translational Modification

Nanopore Discrimination of Coagulation Biomarker Derivatives and Characterization of a Post-Translational Modification

3D Blockage Mapping for Identifying Familial Point Mutations in Single Amyloid‐β Peptides with a Nanopore

Recognition Receptor for Methylated Arginine at the Single Molecular Level

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