Cytosolic proteins begin to fold co-translationally as soon as they emerge from the ribosome during translation. These early co-translational steps are crucial for overall folding and are guided by an intricate network of interactions with molecular chaperones. Because cellular co-translational folding is challenging to detect, its timing and progression remain largely elusive. To quantitatively define co-translational folding in live cells, we developed a high-throughput method that we term “Arrest Peptide Profiling” (AP Profiling). Combining AP Profiling with single-molecule experiments, we delineate co-translational folding for a set of GTPase domains with similar structures, defining how topology shapes folding pathways. Genetic ablation of nascent chain-binding chaperones results in discrete and localized folding changes, highlighting how functional redundancy among chaperones is achieved by distinct engagement with the nascent protein. Our work provides a window into cellular folding pathways of structurally intricate proteins and paves the way for systematic studies of nascent protein folding at exceptional resolution and throughput.

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.