Nanopore Sequencing Breakthrough Maps Full-Length Protein Strands

A new nanopore-based method allows precise single-molecule protein sequencing, which could help provide a clearer understanding of proteoform diversity and post-translational modifications (PTMs). Developed by researchers at the University of Washington, the method combines nanopore technology with the ClpX unfoldase motor to map proteins down to individual amino acid substitutions.

Using a commercial nanopore sensor array, the research team overcame previous limitations in nanopore protein sequencing by ratcheting proteins through a CsgG nanopore with the help of ClpX. The unfoldase pulls proteins through the nanopore in two-residue steps, enabling the detailed mapping of amino acid sequences and PTMs, such as phosphorylation.

The researchers were able to detect sequence variations and enzymatic modifications along protein strands hundreds of amino acids long. The team also explored the rereading capability of individual protein strands, which increases classification accuracy and provides a more robust dataset for identifying proteoforms.

A key innovation of the method is its ability to monitor protein folding and unfolding in real time. By tracking the ionic current changes as proteins pass through the nanopore, the researchers were able to distinguish between folded and unfolded protein domains. This feature is particularly valuable for studying proteins that are difficult to analyze using traditional, primarily mass spectrometry-based methods, such as those with stable tertiary structures.

In addition to single-molecule sensitivity, the team developed a biophysical model to simulate raw nanopore signals, allowing for more accurate interpretation of the sequencing data. The simulation helps link the protein sequence directly to the nanopore current signal, improving the ability to decode complex protein modifications.

The team also applied this method to map phosphorylation sites, an important post-translational modification. Although PTMs are crucial for proteoform diversity, their quantitative analysis, especially in single-molecule contexts, is challenging with the resolution and throughput constraints of current technologies such as mass spectrometry, the researchers noted. By analyzing specific proteins phosphorylated by kinases such as protein kinase A (PKA) and casein kinase II (CKII), the team not only demonstrated the capability to analyze PTMs on whole proteins, but also suggest a pathway towards integrating barcoding with PTM analysis for potential multiplexing – “an avenue yet to be explored,” wrote the researchers.