Single molecule protein sequencing using biological nanopores

Informacje na temat projektu

SMPSBN

Identyfikator umowy o grant: 897672

DOI

10.3030/897672

Projekt został zamknięty

Data podpisania przez KE 1 Kwietnia 2020

Data rozpoczęcia 1 Kwietnia 2020

Data zakończenia 31 Marca 2022

Finansowanie w ramach

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Koszt całkowity

€ 187 572,48

Wkład UE

€ 187 572,48

Koordynowany przez

TECHNISCHE UNIVERSITEIT DELFT
Netherlands

Periodic Reporting for period 1 - SMPSBN (Single molecule protein sequencing using biological nanopores)

Okres sprawozdawczy: 2020-04-01 do 2022-03-31

Podsumowanie kontekstu i ogólnych celów projektu

Biology (and science as a whole) is continually driven forward by advances in experimental technology. Microscopes capable of observing the previously unobservable, analytical techniques allowing us to isolate and study different substances, sequencers providing direct access to the DNA code of life: these developments pave the way to medical, engineering, and scientific discoveries of great importance.

Proteomics remains a relatively dark area in biology, due to the lack of any experimental tool for characterizing samples of protein as small or as heterogeneous as a single cell. The current gold standard in protein identification and sequencing, mass spectrometry, is presently incapable of this task. A tool capable of identifying or sequencing proteins at the single-molecule level would provide us with the capability to answer the seemingly simple yet currently unapproachable question of what, exactly, is floating around in a cell at any given time.

This project's goal was to perform proof-of-principle experiments on a concept for such a technology, based on nanopore sequencing. Nanopore sequencing has additional advantages, as a highly accessible tool with minimal overhead cost and a high degree of engineerability, and as a technology whose core elements are already industrially produced and commercialized. Overall, our hope is to develop a robust single-molecule protein sequencing technology that is broadly accessible to scientists and clinicians.

Prace wykonane od początku projektu do końca okresu sprawozdawczego oraz najważniejsze dotychczasowe rezultaty

Initial set up and infrastructure:
• Constructed a low-noise biological nanopore experimental setup in the Dekker lab at TU Delft.
• Designed and refined a construct for DNA-peptide conjugate strands that allows proteins to be read with a nanopore.
• Expressed and purified Hel308, an helicase ideally suited to use for controlling the DNA handle for nanopore protein sequencing.
• Trained an Erasmus scholar (Foteini Mentzou), a visiting PhD student (Albert Kang), and a Bachelor's student (Theo Koenig) to run experiments and analyze nanopore data, contributing to the educational mission of the university and disseminating expertise and knowledge.

New research and development:
• Investigated strategies for in-house conjugation of DNA and peptides at both N- and C-termini, finding appropriate strategies for both.
• Designed and carried out experiments to measure single-amino-acid substitution variants of three different peptides.
• Identified a strategy for reliably obtaining indefinitely many re-reads of single molecules by increasing helicase concentration.
• Developed a suite of data analysis tools to confidently identify peptide variants from raw nanopore data, and to
• Demonstrated single-read variant identification accuracy on par with early nanopore DNA sequencing experiments, and demonstrated extremely high accuracy using peptide re-reads.
• Collaborated with colleagues at University of Illinois (Prof. Aleksei Aksimentiev and student Jingqian Liu) to show that MD simulations can explain and qualitatively predict ion current signals for the measured peptide sequences.
• Published results of peptide discrimination experiments and MD simulations in Science (Brinkerhoff et al, 2021).

Ongoing work post-publication:
• Continuing to investigate DNA-peptide conjugation strategies in order to apply them to natural peptides directly from biological samples.
• Beginning use of DNA-peptide-DNA triplex constructs, encouraging both capture by the nanopore and helicase control.
• Beginning experiments discriminating phosphorylated from unphosphorylated peptides.
• Designing a next round of experiments:
* re-engineering the MspA pore
* strategies for massively parallel construction of a reference library of DNA-peptide conjugates
* investigating biologically and clinically relevant systems such as MHC immunopeptides
* further improving sample preparation and experimental protocols to increase throughput and quality of data

Innowacyjność oraz oczekiwany potencjalny wpływ (w tym dotychczasowe znaczenie społeczno-gospodarcze i szersze implikacje społeczne projektu)

We accomplished our stated goal of proof-of-principle demonstration for our new single-molecule nanopore protein reader, achieving a previously unseen level of precision in single-molecule identification of proteins. As stated previously, this tool could facilitate a major advance in proteomics if we continue to develop it for application to natural peptides. Much like DNA sequencing from the 1970s onwards, the next revolution in biological science will involve the enormous and variable information content in the proteome, provided we have the tools to access it.

artistic rendering of nanopore protein sequencing scheme