N-Terminal Binding Proteins for Single-Molecule Peptide Sequencing

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Solution Overview

Problem

Current mass spectrometry methods for proteomic characterization face challenges such as quantification difficulties due to varying ionization efficiencies, limited dynamic range, and complications in analyzing phosphopeptides, particularly in complex samples like blood, necessitating improved reagents and methods for sensitive and quantitative analysis of peptides.

Innovation Solution

Development of fluorescently labeled N-terminal amino acid binding proteins (NAABs) and enzymatic Edman degradation methods that allow for single-molecule sequencing of peptides in a neutral aqueous environment, enabling high-throughput analysis with improved dynamic range and specificity for phosphorylated amino acids.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If mass spectrometry is used for proteomic characterization, then high-throughput analysis is achieved, but quantification accuracy deteriorates due to varying ionization efficiencies

Engineering Contradiction:

Improvehigh-throughput analysisVSAvoidquantification accuracy

Core Design Contradiction:

ProductivityVSMeasurement precision

Solution Approach 1:

The patent replaces mass spectrometry-based quantification with a fluorescence-based detection system. Fluorescently labeled N-terminal amino acid binding proteins (NAABs) specifically bind to N-terminal amino acids of peptides, and their binding is detected through fluorescence signals. This substitution eliminates the ionization efficiency variability inherent in mass spectrometry, providing accurate quantification while maintaining high-throughput capability through parallel analysis of multiple peptides.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Speed

If mass spectrometry is used for analyzing complex samples, then analysis speed is improved, but dynamic range deteriorates

Engineering Contradiction:

Improveanalysis speedVSAvoiddynamic range

Core Design Contradiction:

SpeedVSQuantity of substance

Solution Approach 1:

The patent segments the complex sample analysis into discrete, detectable units. Each peptide is analyzed individually through its N-terminal amino acid binding to fluorescently labeled NAABs. This segmentation allows the system to detect and quantify peptides across a wide concentration range without the dynamic range limitations of mass spectrometry, while maintaining rapid analysis speed through parallel processing of multiple peptides.

Inventive Principle:

Principle #1Segmentation

3Measurement precision

If conventional Edman degradation is used, then N-terminal amino acid sequencing is achieved, but phosphopeptide analysis deteriorates due to phosphate loss

Engineering Contradiction:

ImproveN-terminal amino acid sequencing accuracyVSAvoidphosphate loss

Core Design Contradiction:

Measurement precisionVSLoss of substance

Solution Approach 1:

The patent introduces fluorescently labeled N-terminal amino acid binding proteins (NAABs) as intermediary molecules. These NAABs specifically bind to N-terminal amino acids including phosphorylated forms, allowing detection without requiring chemical modification or degradation steps that cause phosphate loss. The NAABs serve as intermediaries that preserve the phosphopeptide structure while enabling specific detection and sequencing.

Inventive Principle:

Principle #24Intermediary (Mediator)

4Productivity

If harsh conditions are used for Edman degradation, then cleavage efficiency is improved, but protein stability deteriorates

Engineering Contradiction:

Improvecleavage efficiencyVSAvoidprotein stability

Core Design Contradiction:

ProductivityVSStability of the object's composition

Solution Approach 1:

The patent changes the operational parameters from harsh chemical conditions to mild physiological conditions. Instead of using strong acids or high temperatures for Edman degradation, the system employs fluorescently labeled NAABs that bind specifically to N-terminal amino acids under neutral conditions. This parameter change maintains protein stability while achieving efficient cleavage and sequencing through the fluorescent binding mechanism.

Inventive Principle:

Principle #35Parameter changes

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 accurate and quantitative sequencing of peptides and phosphorylated forms, enhancing proteomic analysis by providing a complementary method to mass spectrometry with improved sensitivity and dynamic range, suitable for complex biological samples.

Implementation Method 1

fluorescently labeled N-terminal amino acid binding proteins (NAABs)

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Implementation Method 2

specificity for phosphorylated amino acids

Methodology Applied

Scientific EffectMolecular recognition:

Implementation Method 3

enzymatic Edman degradation methods

Methodology Applied

Scientific EffectEnzyme catalysis: Enzyme

Implementation Method 4

cleaving the N-terminal amino acid of the polypeptide

Methodology Applied

Scientific EffectHydrolysis: Hydrolysis

Data Source

PatentUS12493042B2Molecules and methods for iterative polypeptide analysis and processing

Publication Date: 2025.12.09 WASHINGTON UNIV IN SAINT LOUIS

AI summary

Reagents and methods for the digital analysis of proteins or peptides are provided. Specifically provided herein are proteins for identifying the N-terminal amino acid or N-terminal phosphorylated amino acid of a polypeptide. Also, an enzyme for use in the cleavage step of the Edman degradation reaction and a method for using this enzyme are described.