High-Throughput Human Histone Detection by an Engineered Actinoporin Nanopore

Area of Science:

• Biomolecular engineering
• Nanotechnology
• Biosensing

Background:

• Protein nanopores show promise for protein identification and peptide sequencing.
• Complex protein properties hinder their widespread use in biosensor applications compared to nucleic acids.

Purpose of the Study:

• To develop an engineered α-helical Fav nanopore for detecting human histones.
• To utilize a commercial MinION device for high-throughput protein analysis.
• To enable discrimination between different histone variants and post-translationally modified forms.

Main Methods:

• Development of an α-helical Fav nanopore from Orbicella faveolata.
• Stable insertion of engineered nanopores into a polymeric membrane.
• Bulk analysis of nanopore current and noise for histone capture.
• Detailed blocking analyses for histone-specific signal identification.

Main Results:

• Engineered Fav nanopores demonstrated stable membrane insertion.
• Significant differences in nanopore current and noise were observed for different human histones.
• Histone-specific signals allowed discrimination between medically important extracellular histones in mixtures.
• The approach enabled detection of full-length human histones and their post-translationally modified variants.

Conclusions:

• The developed Fav nanopore provides a novel tool for protein detection.
• This high-throughput nanopore approach facilitates the analysis of biomedically important proteins.
• The study represents a significant advancement towards the broader application of nanopores in analytics.