Detection of Phosphorylation Post-translational Modifications Along Single Peptides with Nanopores

Overview

Journal Nat Biotechnol

Specialty Biotechnology

Date 2023 Jun 29

PMID 37386295

Authors

Ian C Nova

Justas Ritmejeris

Henry Brinkerhoff

Theo J R Koenig

Jens H Gundlach

Cees Dekker

Affiliations

Soon will be listed here.

Abstract

Current methods to detect post-translational modifications of proteins, such as phosphate groups, cannot measure single molecules or differentiate between closely spaced phosphorylation sites. We detect post-translational modifications at the single-molecule level on immunopeptide sequences with cancer-associated phosphate variants by controllably drawing the peptide through the sensing region of a nanopore. We discriminate peptide sequences with one or two closely spaced phosphates with 95% accuracy for individual reads of single molecules.

Citing Articles

Electrokinetic detection of single-molecule phosphorylation.

Wang Q bioRxiv. 2025; .

PMID: 40060633 PMC: 11888256. DOI: 10.1101/2025.02.24.640009.

Bio-nanopore technology for biomolecules detection.

Li P, Liang D, Yang E, Zeb M, Huang H, Sun H Adv Biotechnol (Singap). 2025; 2(4):45.

PMID: 39883334 PMC: 11740844. DOI: 10.1007/s44307-024-00051-7.

Genetic Code Expansion: Recent Developments and Emerging Applications.

Huang Y, Zhang P, Wang H, Chen Y, Liu T, Luo X Chem Rev. 2024; 125(2):523-598.

PMID: 39737807 PMC: 11758808. DOI: 10.1021/acs.chemrev.4c00216.

Hetero-Oligomeric Protein Pores for Single-Molecule Sensing.

Satheesan R, Janeena A, Mahendran K J Membr Biol. 2024; .

PMID: 39699641 DOI: 10.1007/s00232-024-00331-2.

Successful adaptation of a MinION nanopore for protein sequencing.

Berezin C Synth Biol (Oxf). 2024; 9(1):ysae017.

PMID: 39605954 PMC: 11602029. DOI: 10.1093/synbio/ysae017.

References

Kim M, Zhong J, Pandey A . Common errors in mass spectrometry-based analysis of post-translational modifications. Proteomics. 2015; 16(5):700-14. PMC: 5548100. DOI: 10.1002/pmic.201500355. View

Xu H, Wang Y, Lin S, Deng W, Peng D, Cui Q . PTMD: A Database of Human Disease-associated Post-translational Modifications. Genomics Proteomics Bioinformatics. 2018; 16(4):244-251. PMC: 6205080. DOI: 10.1016/j.gpb.2018.06.004. View

Engelhard V, Obeng R, Cummings K, Petroni G, Ambakhutwala A, Chianese-Bullock K . MHC-restricted phosphopeptide antigens: preclinical validation and first-in-humans clinical trial in participants with high-risk melanoma. J Immunother Cancer. 2020; 8(1). PMC: 7228659. DOI: 10.1136/jitc-2019-000262. View

Rosen C, Rodriguez-Larrea D, Bayley H . Single-molecule site-specific detection of protein phosphorylation with a nanopore. Nat Biotechnol. 2014; 32(2):179-81. PMC: 4391620. DOI: 10.1038/nbt.2799. View

Restrepo-Perez L, Wong C, Maglia G, Dekker C, Joo C . Label-Free Detection of Post-translational Modifications with a Nanopore. Nano Lett. 2019; 19(11):7957-7964. PMC: 6856961. DOI: 10.1021/acs.nanolett.9b03134. View

Wloka C, Van Meervelt V, van Gelder D, Danda N, Jager N, Williams C . Label-Free and Real-Time Detection of Protein Ubiquitination with a Biological Nanopore. ACS Nano. 2017; 11(5):4387-4394. PMC: 5444049. DOI: 10.1021/acsnano.6b07760. View

Shorkey S, Du J, Pham R, Strieter E, Chen M . Real-Time and Label-Free Measurement of Deubiquitinase Activity with a MspA Nanopore. Chembiochem. 2021; 22(17):2688-2692. PMC: 8416795. DOI: 10.1002/cbic.202100092. View

Nir I, Huttner D, Meller A . Direct Sensing and Discrimination among Ubiquitin and Ubiquitin Chains Using Solid-State Nanopores. Biophys J. 2015; 108(9):2340-9. PMC: 4423055. DOI: 10.1016/j.bpj.2015.03.025. View

Fahie M, Chen M . Electrostatic Interactions between OmpG Nanopore and Analyte Protein Surface Can Distinguish between Glycosylated Isoforms. J Phys Chem B. 2015; 119(32):10198-206. PMC: 4958047. DOI: 10.1021/acs.jpcb.5b06435. View

10.

Abraham Versloot R, Lucas F, Yakovlieva L, Tadema M, Zhang Y, Wood T . Quantification of Protein Glycosylation Using Nanopores. Nano Lett. 2022; 22(13):5357-5364. PMC: 9284675. DOI: 10.1021/acs.nanolett.2c01338. View

11.

Brinkerhoff H, Kang A, Liu J, Aksimentiev A, Dekker C . Multiple rereads of single proteins at single-amino acid resolution using nanopores. Science. 2021; 374(6574):1509-1513. PMC: 8811723. DOI: 10.1126/science.abl4381. View

12.

Yan S, Zhang J, Wang Y, Guo W, Zhang S, Liu Y . Single Molecule Ratcheting Motion of Peptides in a Porin A (MspA) Nanopore. Nano Lett. 2021; 21(15):6703-6710. DOI: 10.1021/acs.nanolett.1c02371. View

13.

Chen Z, Wang Z, Xu Y, Zhang X, Tian B, Bai J . Controlled movement of ssDNA conjugated peptide through porin A (MspA) nanopore by a helicase motor for peptide sequencing application. Chem Sci. 2022; 12(47):15750-15756. PMC: 8654031. DOI: 10.1039/d1sc04342k. View

14.

Butler T, Pavlenok M, Derrington I, Niederweis M, Gundlach J . Single-molecule DNA detection with an engineered MspA protein nanopore. Proc Natl Acad Sci U S A. 2008; 105(52):20647-52. PMC: 2634888. DOI: 10.1073/pnas.0807514106. View

15.

Derrington I, Craig J, Stava E, Laszlo A, Ross B, Brinkerhoff H . Subangstrom single-molecule measurements of motor proteins using a nanopore. Nat Biotechnol. 2015; 33(10):1073-5. PMC: 4915380. DOI: 10.1038/nbt.3357. View

16.

Manrao E, Derrington I, Laszlo A, Langford K, Hopper M, Gillgren N . Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase. Nat Biotechnol. 2012; 30(4):349-53. PMC: 3757088. DOI: 10.1038/nbt.2171. View

17.

Cherf G, Lieberman K, Rashid H, Lam C, Karplus K, Akeson M . Automated forward and reverse ratcheting of DNA in a nanopore at 5-Å precision. Nat Biotechnol. 2012; 30(4):344-8. PMC: 3408072. DOI: 10.1038/nbt.2147. View

18.

Laszlo A, Derrington I, Brinkerhoff H, Langford K, Nova I, Samson J . Detection and mapping of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore MspA. Proc Natl Acad Sci U S A. 2013; 110(47):18904-9. PMC: 3839702. DOI: 10.1073/pnas.1310240110. View

19.

Laszlo A, Derrington I, Ross B, Brinkerhoff H, Adey A, Nova I . Decoding long nanopore sequencing reads of natural DNA. Nat Biotechnol. 2014; 32(8):829-33. PMC: 4126851. DOI: 10.1038/nbt.2950. View

20.

Requiao R, Fernandes L, de Souza H, Rossetto S, Domitrovic T, Palhano F . Protein charge distribution in proteomes and its impact on translation. PLoS Comput Biol. 2017; 13(5):e1005549. PMC: 5460897. DOI: 10.1371/journal.pcbi.1005549. View