Peptide Substrates for BoNT/E Detection via Mass Spectrometry

Solution Overview

Problem

Current methods for detecting botulinum neurotoxin type E (BoNT/E) are either insensitive, time-consuming, or require animal sacrifice, making them inadequate for rapid and sensitive detection and differentiation.

Innovation Solution

Development of novel peptide substrates selectively recognized by BoNT/E, combined with mass spectrometry for proteolytic cleavage analysis, allowing for sensitive detection and quantification of active toxin in samples.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If the mouse bioassay is used for detecting botulinum toxin, then sensitivity is improved (detecting as little as 10 pg of active toxin), but time consumption increases (taking up to 4 days for final results) and animal sacrifice is required

Engineering Contradiction:

Improvedetection sensitivityVSAvoidassay duration

Core Design Contradiction:

Measurement precisionVSLoss of time

Solution Approach 1:

The patent replaces the biological mouse bioassay system with a chemical/physical mass spectrometry system. The detection method substitutes animal-based biological assays with MS-based proteolytic cleavage analysis, using peptide substrates that are cleaved by BoNT/E and detected by mass spectrometry, thereby eliminating the need for animal sacrifice while maintaining high sensitivity

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The patent changes the detection parameter from measuring mortality symptoms in mice to measuring mass-to-charge ratio of peptide cleavage products. By monitoring the specific mass signature of cleaved peptide substrates, the method achieves rapid detection (within hours rather than days) while maintaining the ability to detect very low toxin concentrations

Inventive Principle:

Principle #35Parameter changes

2Measurement precision

If the mouse bioassay is used for detecting botulinum toxin, then detection sensitivity is improved, but the number of animals required increases

Engineering Contradiction:

Improvedetection sensitivityVSAvoidnumber of animals

Core Design Contradiction:

Measurement precisionVSQuantity of substance

Solution Approach 1:

The patent replaces the biological mouse bioassay system with a chemical/physical mass spectrometry system. The detection method substitutes animal-based biological assays with MS-based proteolytic cleavage analysis, using peptide substrates that are cleaved by BoNT/E and detected by mass spectrometry, thereby eliminating the need for animal sacrifice while maintaining high sensitivity

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

3Productivity

If rapid detection methods are developed to reduce time consumption, then assay speed is improved, but detection sensitivity may deteriorate

Engineering Contradiction:

Improvedetection speedVSAvoiddetection sensitivity

Core Design Contradiction:

ProductivityVSMeasurement precision

Solution Approach 1:

The patent performs preliminary enrichment of the toxin by incubating the sample with peptide substrates before mass spectrometry analysis. This pre-incubation step allows the toxin to bind to and cleave the peptide substrates, concentrating the detection signal and enabling sensitive detection even when the toxin is present at very low concentrations, while still achieving rapid results within hours

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The patent replaces the biological mouse bioassay system with a chemical/physical mass spectrometry system. The detection method substitutes animal-based biological assays with MS-based proteolytic cleavage analysis, using peptide substrates that are cleaved by BoNT/E and detected by mass spectrometry, thereby eliminating the need for animal sacrifice while maintaining high sensitivity

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

Enables rapid and sensitive detection of BoNT/E at low concentrations, reducing the need for animal testing and improving diagnostic efficiency.

Implementation Method 1

The botulinum toxins belong to the family of zinc metallopeptidases and more particularly to the sub-family of the zincins which contain the consensus sequence HExxH; They cleave very specifically the neuronal proteins involved in the exocytosis of the neurotransmitters

Methodology Applied

Scientific EffectProteolytic cleavage: Hydrolysis

Implementation Method 2

Development of novel peptide substrates selectively recognized by BoNT/E, combined with mass spectrometry for proteolytic cleavage analysis, allowing for sensitive detection and quantification of active toxin in samples

Methodology Applied

Scientific EffectMass spectrometry:

Data Source

PatentUS10408837B2Peptide substrates recognizable by type E botulinum neurotoxin

Publication Date: 2019.09.10 THE GOVERNMENT OF THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY DEPARTMENT OF HEALTH & HUMAN SERVICES

AI summary

The present invention relates to peptide substrates selectively recognized by botulinum toxin type A, BoNT/E, and their uses, in particular for carrying out methods for detecting, identifying and/or diagnosing botulinum toxin type E.