Maglia and colleagues discuss advances in nanopore technology en route to single-molecule protein sequencing

This protocol outlines a cost-effective and rapid protein sample pre-fractionation strategy based on highly efficient passive elution from gels, recovering intact proteins for top-down proteomics or partially digested proteins for middle-down proteomics.

The antibody response to infection and vaccination is an essential component of the anti-infective immune response. Here the authors present a de novo protein sequencing method for antibody discovery from polyclonal IgG from human plasma and characterise the antibody response to the Moderna Spikevax COVID-19 vaccine.

A technique for threading long protein strands through a nanopore by electrophoresis and back using a protein unfoldase motor, ClpX, enables single protein molecules to be analyzed multiple times with single-amino-acid sensitivity.

Understanding protein function is vital for biomedicine. Here, authors develop a method using statistics-informed graph networks to predict functions from sequences. The method integrates evolutionary couplings and residue communities to improve the accuracy of function annotations for proteins.

Homologous protein search is one of the most commonly used methods for protein analysis. Here, authors propose PLMSearch, a search method that takes only sequences as input and can search millions of protein pairs in seconds while maintaining sensitivity comparable to SOTA structure search methods.

Ancient proteins can provide phylogenetic information at a timescale that supersedes ancient DNA. Paleoproteomics could thus provide invaluable evolutionary insights, including into human evolution.

Developments in nanopore-based peptide detection and sequencing show promise of a breakthrough.

By using fixed charges to engineer a strong electroosmotic flow, we achieve the unidirectional transport of natural polypeptides across nanopores. Our approach enables native proteins to be transported enzymatically and non-enzymatically in the absence of denaturant and electrophoretic tags, with potential applications for protein sequencing.

The nanopore community is stepping toward a new frontier of single-molecule protein sequencing. Here, we offer our opinions on the unique potential for this emerging technology, with a focus on single-cell proteomics, and some challenges that must be overcome to realize it.