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.

Increasing Fluid Viscosity Ensures Consistent Single-Cell Encapsulation.

High-throughput single-cell analysis typically relies on the isolation of cells of interest in separate compartments for subsequent phenotypic or genotypic characterization. Using microfluidics, this is achieved by isolating individual cells in microdroplets or microwells. However, due to cell-to-cell variability in size, shape, and density, the cell capture efficiencies may vary significantly. This variability can negatively impact the measurements and introduce undesirable artifacts when trying to isolate and characterize heterogeneous cell populations. In this study, we show that single-cell isolation biases in microfluidics can be circumvented by increasing the viscosity of fluids in which cells are dispersed. At a viscosity of 40-50 cP (cP), the cell sedimentation is effectively reduced, resulting in a steady cell flow inside the microfluidics chip and consistent encapsulation in water-in-oil droplets over extended periods of time. This approach allows nearly all cells in a sample to be isolated with the same efficiency, irrespective of their type. Our results show that increased fluid viscosity, rather than cell-adjusted density, provides a more reliable approach to mitigate single-cell isolation biases.

Detection of phosphorylation post-translational modifications along single peptides with nanopores.

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.