In this thesis, I present the development of biophysical and chemical strategies to enable single-molecule protein sequencing using biological nanopores. While genomic sequencing has transformed biology by enabling the massive-scale amplification and sequencing of nucleic acids, a comparable revolution in proteomics has remained elusive. Proteins cannot be amplified, and existing techniques – most prominently mass spectrometry – rely on ensemble averaging, destroying labile modifications and masking molecular heterogeneity. The aim of this thesis was to develop a framework for direct amplification-free analysis of individual peptides using nanopore-based sequencing....

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.

Protein sequencing and the identification of post-translational modifications are key to understanding cellular signalling pathways and metabolic processes in health and disease. Nanopores, that is, nanometre-sized holes in a membrane, were previously put to use for DNA and RNA sequencing, offering single-molecule sensitivity and long read lengths. This prompted efforts to engineer nanopores for the high-throughput sequencing of peptides and proteins. In this Review, we discuss research towards single-molecule protein sequencing using biological nanopores, investigating how their sensitivity allows the discrimination of all 20 amino acids. We outline how fingerprinting of proteins is facilitated by using motor proteins and electro-osmotic flow to promote the slow translocation of proteins through nanopores. Moreover, we examine applications of nanopores to the identification of post-translational modifications, highlighting the potential of this technology for fundamental and clinical proteomic studies. Finally, we outline the advantages and limitations of nanopore systems for protein sequencing and the challenges that remain to be overcome for realizing de novo nanopore protein sequencing.

Peptide hormones are decorated with post-translational modifications (PTMs) that are crucial for receptor recognition. Tyrosine sulfation on plant peptide hormones is, for example, essential for plant growth and development. Measuring the occurrence and position of sulfotyrosine is, however, compromised by major technical challenges during isolation and detection. Nanopores can sensitively detect protein PTMs at the single-molecule level. By translocating PTM variants of the plant pentapeptide hormone phytosulfokine (PSK) through a nanopore, we here demonstrate the 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 powerful tool for identifying position-specific sulfation on peptide hormones and promising wider applications to identify protein PTMs.

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.