Biology subjects

Albada, B.

Publications and source records attributed to Albada, B..

3 recordsLinked to original sources

bioRxiv2025

Terminal conjugation enables nanopore sequencing of peptides

Nanopore sequencing of peptides holds great promise for single-molecule proteomics, but robust conjugation strategies to adapt native peptides for motor-enzyme-driven translocation have yet to be developed. Here, we establish terminally directed DNA-peptide conjugation chemistry strategies that expand the applicability of nanopore sequencing beyond synthetic model systems to natural peptides. At the N terminus, omniligase catalyzes rapid and peptide ligation of a DNA handle under mild conditions. At the C terminus, photoredox decarboxylative ligation introduces a bioorthogonal linker that enables CuAAC-mediated DNA attachment that ensures proper stretching and translocation of short peptides through the nanopore. Our study reveals that long peptides can be sequenced with single-end conjugation, while short or neutral peptides require threading tails. Positively charged peptides cannot be translocated under the same electric field but can be sequenced after charge neutralization. The data demonstrate controlled nanopore readouts of peptides that differ widely in length, charge, and sequence. This framework establishes a versatile chemical foundation for adapting natural peptides to nanopore sequencing, advancing single-molecule proteomic analysis.

biophysics↗

bioRxiv2024

Resolving sulfation PTMs on a plant peptide hormone using nanopore sequencing

Peptide phytohormones are decorated with post-translational modifications (PTMs) that are crucial for receptor recognition. Tyrosine sulfation on these hormones is essential for plant growth and development1. Measuring the occurrence and position of sulfotyrosine is, however, compromised by major technical challenges during isolation and detection2. We recently introduced a nanopore peptide sequencing method that sensitively detects PTMs at the single-molecule level3. By translocating PTM variants of the plant pentapeptide hormone phytosulfokine (PSK) through a nanopore, we here demonstrate accurate identification of sulfation and phosphorylation on the two tyrosine residues of PSK. Sulfation can be clearly detected and distinguished (>90%) from phosphorylation on the same residue. Moreover, the presence or absence of PTMs on the two close-by tyrosine residues can be accurately determined (>96% accuracy). Our findings demonstrate the extraordinary sensitivity of nanopore protein measurements, providing a new tool for identifying sulfation on peptide phytohormones and promising wider applications to identify protein PTMs.

biophysics↗

bioRxiv2023

Rapid Molecular Mechanotyping with Microfluidic Force Spectroscopy

Molecular mechanotyping, the quantification of changes in the stability of supramolecular interactions and chemical bonds under the action of mechanical forces, is an essential tool in the field of mechanochemistry. This is conventionally done in single-molecule force-spectroscopy (smFS) assays, for example with optical tweezers or Atomic Force Microscopy. While these techniques provide detailed mechanochemical insights, they are time-consuming, technically demanding and expensive; as a result, high-throughput screening of the mechanochemical properties of molecules of interest is challenging. To resolve this, we present a rapid, simple and low-cost mechanotyping assay: microfluidic force spectroscopy ({micro}FFS), which probes force-dependent bond stability by measuring the detachment of microparticles, bound to microfluidic channels by the interaction of interest, under hydrodynamic forcing. As this allows the simultaneous observation of hundreds of microparticles, we obtain a quantitative mechanotype in a single measurement, using readily available equipment. We validate our method by studying the stability of DNA duplexes, previously characterized through smFS. We further show that we can quantitatively describe the experimental data with simulations, which allows us to link the {micro}FFS data to single-bond mechanochemical properties. This opens the way to use ({micro}FFS) as a rapid molecular mechanotyping tool.

biophysics↗