Enzyme-less nanopore detection of post-translational modifications within long polypeptides

Area of Science:

• Biophysics
• Proteomics
• Molecular Biology

Background:

• Analyzing cellular proteins and their variants at the single-molecule level is crucial for biological discovery.
• Nanopore technology offers potential for full-length proteoform identification.

Purpose of the Study:

• To develop a method for analyzing individual polypeptides at the single-molecule level.
• To demonstrate the capability of nanopore technology for proteoform identification and post-translational modification mapping.

Main Methods:

• Utilizing electro-osmosis in a charge-selective nanopore for non-enzymatic capture, unfolding, and translocation of long polypeptides (>1,200 residues).
• Monitoring ionic current changes during polypeptide translocation to detect modifications.
• Employing chaotropic reagents at non-denaturing concentrations to accelerate analysis.

Main Results:

• Demonstrated directional, co-translocational unfolding of unlabelled thioredoxin polyproteins through the nanopore.
• Successfully located post-translational modifications within polypeptide chains by analyzing ionic current.
• Established a foundation for creating comprehensive proteoform inventories.

Conclusions:

• Nanopore technology can analyze individual proteins and their modifications at the single-molecule level.
• This approach facilitates the identification and characterization of diverse proteoforms.
• The method paves the way for detailed proteomic analysis in biological samples.