Mass Spectrometry Analysis of Covalent Probe-Bound Proteins

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Solution Overview

Problem

Current methods for identifying proteins or biomolecules covalently modified by small molecule probes are challenging due to biochemical complexity and dynamic range of mammalian proteomes, leading to high or indeterminate false-positive/negative rates, especially when determining the fraction of endogenous target molecules bound by covalent probes.

Innovation Solution

An analytical method involving contacting a test compound with a polypeptide to form a test compound-polypeptide conjugate, analyzing using mass spectrometry, detecting thiolated ions, and identifying irreversible bonding based on mass spectrometry assay results, which includes specific solvent conditions, enzyme treatments, and isotopic labeling for accurate stoichiometry measurement.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If mass spectrometry-based identification is used to detect covalently modified proteins, then identification capability is provided, but high false-positive/negative rates occur due to biochemical complexity and dynamic range of mammalian proteomes

Engineering Contradiction:

Improveidentification accuracyVSAvoidfalse-positive/negative rate

Core Design Contradiction:

Measurement precisionVSReliability

Solution Approach 1:

The patent introduces an affinity tag (such as biotin) or biorthogonal reactive group (such as alkyne) as an intermediary 'handle' that mediates between the covalent probe and the detection system. This handle enables specific enrichment of probe-bound proteins from complex biological mixtures through affinity purification, thereby improving measurement precision and reducing false positives caused by the complexity of mammalian proteomes

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent segments the detection process into distinct stages: (1) covalent probe binding to target proteins, (2) affinity tag-mediated enrichment of bound proteins, and (3) mass spectrometry identification. This segmentation allows each stage to be optimized independently, improving overall reliability by isolating the detection step from the complexity of the full proteome

Inventive Principle:

Principle #1Segmentation

2Quantity of substance

If affinity tags or biorthogonal reactive groups are added to small molecule probes, then enrichment capability is improved, but binding kinetics and activity are disrupted and cell plasma membrane permeability is negated

Engineering Contradiction:

Improveenrichment efficiencyVSAvoidbinding activity

Core Design Contradiction:

Quantity of substanceVSReliability

Solution Approach 1:

The patent applies preliminary action by performing the covalent binding step first in live cells using the native probe structure, then subsequently adding the affinity tag-containing reagent to capture the already-bound complexes. This sequence preserves the natural binding kinetics and membrane permeability of the original probe while still enabling enrichment

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The affinity tag serves as an intermediary that binds to the probe-target complex without interfering with the original probe-target interaction. The tag provides the enrichment capability while the native probe maintains its binding activity and cellular permeability

Inventive Principle:

Principle #24Intermediary (Mediator)

3Measurement precision

If indirect competition format with broad-activity probes is used, then target identification is enabled, but enrichment capability is lost and stochastic/abundance-biased sequencing limitations remain

Engineering Contradiction:

Improvetarget identificationVSAvoidenrichment efficiency

Core Design Contradiction:

Measurement precisionVSProductivity

Solution Approach 1:

The patent introduces an affinity tag as an intermediary that enables direct enrichment of probe-bound proteins, replacing the indirect competition format. This mediator allows specific capture of target proteins through affinity purification, overcoming the stochastic and abundance-biased limitations of indirect methods while maintaining target identification capability

Inventive Principle:

Principle #24Intermediary (Mediator)

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

This method provides a high-throughput, accurate identification of test compounds irreversibly binding to polypeptides, reducing false-positive/negative rates and enabling precise characterization of covalent probe-target interactions.

Implementation Method 1

analyzing the test compound-polypeptide conjugate using a mass spectrometry assay; detecting one or more thiolated ions, or derivative ions thereof, produced in the mass spectrometry assay

Methodology Applied

Scientific EffectMass spectrometry:

Data Source

PatentUS11789025B2Reagents and methods for analysis of proteins and metabolites targeted by covalent probes

Publication Date: 2023.10.17 DANA FARBER CANCER INSTITUTE INC

AI summary

The present application relates to mass spectrometry methods for use in identifying proteins or other biomolecules which are bound irreversibly by test compounds.