Enzyme-less nanopore detection of post-translational modifications within long polypeptides

Nature Nanotechnology, Год журнала: 2023, Номер 18(11), С. 1335 - 1340

Опубликована: Июль 27, 2023

Means to analyse cellular proteins and their millions of variants at the single-molecule level would uncover substantial information previously unknown biology. Nanopore technology, which underpins long-read DNA RNA sequencing, holds potential for full-length proteoform identification. We use electro-osmosis in an engineered charge-selective nanopore non-enzymatic capture, unfolding translocation individual polypeptides more than 1,200 residues. Unlabelled thioredoxin polyproteins undergo transport through nanopore, with directional co-translocational occurring unit by from either C or N terminus. Chaotropic reagents non-denaturing concentrations accelerate analysis. By monitoring ionic current flowing we locate post-translational modifications deep within polypeptide chains, laying groundwork compiling inventories proteoforms cells tissues.

Язык: Английский

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Translocation of linearized full-length proteins through an engineered nanopore under opposing electrophoretic force

Nature Biotechnology, Год журнала: 2023, Номер 42(8), С. 1275 - 1281

Опубликована: Сен. 18, 2023

Язык: Английский

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Real-time detection of 20 amino acids and discrimination of pathologically relevant peptides with functionalized nanopore

Nature Methods, Год журнала: 2024, Номер 21(4), С. 609 - 618

Опубликована: Март 5, 2024

Abstract Precise identification and quantification of amino acids is crucial for many biological applications. Here we report a copper(II)-functionalized Mycobacterium smegmatis porin A (MspA) nanopore with the N91H substitution, which enables direct all 20 proteinogenic when combined machine-learning algorithm. The validation accuracy reaches 99.1%, 30.9% signal recovery. feasibility ultrasensitive was also demonstrated at nanomolar range. Furthermore, capability this system real-time analyses two representative post-translational modifications (PTMs), one unnatural acid ten synthetic peptides using exopeptidases, including clinically relevant associated Alzheimer’s disease cancer neoantigens, demonstrated. Notably, our strategy successfully distinguishes only difference from hydrolysate provides possibility to infer peptide sequence.

Язык: Английский

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Nanopore DNA sequencing technologies and their applications towards single-molecule proteomics

Nature Chemistry, Год журнала: 2024, Номер 16(3), С. 314 - 334

Опубликована: Март 1, 2024

Язык: Английский

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Multi-pass, single-molecule nanopore reading of long protein strands

Nature, Год журнала: 2024, Номер 633(8030), С. 662 - 669

Опубликована: Сен. 11, 2024

Язык: Английский

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Engineering Biological Nanopore Approaches toward Protein Sequencing

ACS Nano, Год журнала: 2023, Номер 17(17), С. 16369 - 16395

Опубликована: Июль 25, 2023

Biotechnological innovations have vastly improved the capacity to perform large-scale protein studies, while methods we for identifying and quantifying individual proteins are still inadequate sequencing at single-molecule level. Nanopore-inspired systems devoted understanding how single molecules behave been extensively developed applications in genome sequencing. These nanopore emerging as prominent tools identification, detection, analysis, suggesting realistic prospects novel This review summarizes recent advances biological sensors toward sequencing, from identification of amino acids controlled translocation peptides proteins, with attention focused on device algorithm development delineation molecular mechanisms aid simulations. Specifically, aims offer recommendations advancement nanopore-based an engineering perspective, highlighting need collaborative efforts across multiple disciplines. should include chemical conjugation, engineering, simulation, machine-learning-assisted electronic fabrication enable practical implementation real-world scenarios.

Язык: Английский

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Deep Learning-Assisted Single-Molecule Detection of Protein Post-translational Modifications with a Biological Nanopore

ACS Nano, Год журнала: 2023, Номер 18(2), С. 1504 - 1515

Опубликована: Дек. 19, 2023

Protein post-translational modifications (PTMs) play a crucial role in countless biological processes, profoundly modulating protein properties on both spatial and temporal scales. PTMs have also emerged as reliable biomarkers for several diseases. However, only handful of techniques are available to accurately measure their levels, capture complexity at single molecule level, characterize multifaceted roles health disease. Nanopore sensing provides high sensitivity the detection low-abundance proteins, holding potential impact single-molecule proteomics PTM detection, particular. Here, we demonstrate ability nanopore, pore-forming toxin aerolysin, detect distinguish α-synuclein-derived peptides bearing or multiple PTMs, namely, phosphorylation, nitration, oxidation occurring different positions various combinations. The characteristic current signatures α-synuclein peptide its variants could be confidently identified by using deep learning model signal processing. We further that this framework can quantify picomolar concentrations C-terminal generated digestion full-length α-synuclein. Collectively, our work highlights advantage nanopores tool simultaneous facilitates use biomarker discovery diagnostics.

Язык: Английский

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Not if but when nanopore protein sequencing meets single-cell proteomics

Nature Methods, Год журнала: 2023, Номер 20(3), С. 336 - 338

Опубликована: Март 1, 2023

Язык: Английский

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Phospholipid Membranes as Chemically and Functionally Tunable Materials

Advanced Materials, Год журнала: 2024, Номер 36(23)

Опубликована: Март 8, 2024

Abstract The sheet‐like lipid bilayer is the fundamental structural component of all cell membranes. Its building blocks are phospholipids and cholesterol. Their amphiphilic structure spontaneously leads to formation a in aqueous environment. Lipids not just elements. Individual species, membrane structure, dynamics influence regulate protein function. An exciting field emerging where membrane‐associated material properties different systems used designing innovative solutions for widespread applications across various fields, such as food industry, cosmetics, nano‐ biomedicine, drug storage delivery, biotechnology, biosensors, computing. Here, authors summarize what known about how lipids determine functions biological membranes this has been or can be translated into applications. Based on recent progress understanding dynamics, physical properties, perspective provided membrane‐controlled regulation extend current even offer new

Язык: Английский

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KSHV 3.0: a state-of-the-art annotation of the Kaposi’s sarcoma-associated herpesvirus transcriptome using cross-platform sequencing

mSystems, Год журнала: 2024, Номер unknown

Опубликована: Янв. 11, 2024

Kaposi's sarcoma-associated herpesvirus (KSHV) is a large, oncogenic DNA virus belonging to the gammaherpesvirus subfamily. KSHV has been extensively studied with various high-throughput RNA-sequencing approaches map transcription start and end sites, splice junctions, translation initiation sites. Despite these efforts, comprehensive annotation of viral transcriptome remains incomplete. In present study, we generated long-read sequencing data set lytic latent using native RNA direct cDNA-sequencing methods. This was supplemented Cap Analysis Gene Expression based on short-read platform. We also utilized sets from previous publications for our analysis. As result this combined approach, have identified number novel transcripts isoforms either corroborated or improved previously molecules, thereby notably enhancing comprehension transcriptomic architecture genome. evaluated coding capability thought be non-coding by integrating translatomic information other publications.IMPORTANCEDeciphering great importance because can gain insight into molecular mechanism replication pathogenesis, which help develop potential targets antiviral interventions. Specifically, identification substantial transcriptional overlaps work suggests existence genome-wide interference between machineries. finding indicates presence regulatory layer, potentially controlling expression genes.

Язык: Английский

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