Elemental Analysis Kinase Assay Kit Multiplexing

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Solution Overview

Problem

Current methods for evaluating protein post-translational modifications, such as phosphorylation and dephosphorylation, face limitations including low sensitivity, difficulty in multiplexing, and limitations in ATP concentration, making it challenging to develop a coherent screening-profiling platform for kinase and phosphatase assays.

Innovation Solution

The development of a method involving elemental analysis to detect and measure protein kinase and phosphatase activity using ATP, kinases, and substrates labeled with element tags, allowing for the separation and quantification of phosphorylated substrates, enabling high-throughput assays that can be applied to various types of kinases and phosphatases in different conditions.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If traditional platform technologies (Radioactivity, Fluorescence Polarization, Time Resolved Fluorescence, FRET) are used for kinase/phosphatase assays, then the assays can be performed with existing methods, but the sensitivity is low and multiplexing capability is limited

Engineering Contradiction:

ImprovesensitivityVSAvoidmultiplexing capability

Core Design Contradiction:

Measurement precisionVSAdaptability or versatility

Solution Approach 1:

The patent replaces traditional optical detection systems (fluorescence, FRET) with mass spectrometry-based detection. This substitution enables simultaneous detection of multiple kinases/phosphatases through mass-to-charge ratio differentiation, achieving both high sensitivity and multiplexing capability without the limitations of optical signal interference

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The invention creates a universal assay platform using standardized peptide substrates with universal tags that can be detected by mass spectrometry. This single platform can assess activity of multiple different kinases and phosphatases simultaneously, replacing the need for multiple specialized assay systems

Inventive Principle:

Principle #6Universality (Multi-functionality)

2Adaptability or versatility

If heterogeneous assay systems are used for different kinases/phosphatases, then various enzyme types can be tested, but the system complexity increases and coherence for high-throughput screening is lost

Engineering Contradiction:

Improveenzyme type coverageVSAvoidassay system coherence

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The patent employs universal tagged peptide substrates that maintain consistent structural features across different kinase and phosphatase assays. This universal design allows all assays to be performed in the same mass spectrometry-based system with standardized protocols, achieving both broad enzyme coverage and system coherence for high-throughput screening

Inventive Principle:

Principle #6Universality (Multi-functionality)

3Quantity of substance

If ATP concentration is limited in the assay system, then the kinase reaction can proceed, but the sensitivity and detection capability are reduced

Engineering Contradiction:

ImproveATP concentrationVSAvoiddetection capability

Core Design Contradiction:

Quantity of substanceVSMeasurement precision

Solution Approach 1:

The patent replaces traditional optical detection methods with mass spectrometry detection, which can detect phosphorylated peptides at very low ATP concentrations. The mass spectrometry system's high sensitivity allows accurate measurement even when ATP is limiting, as detection is based on mass-to-charge ratio rather than optical signal intensity that requires higher concentrations

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

This approach provides a sensitive, robust, and quantitative assay for protein post-translational modifications, enabling the simultaneous evaluation of multiple kinases and phosphatases in cell-free systems and cellular lysates, overcoming the limitations of existing technologies by enhancing sensitivity and multiplexing capabilities.

Implementation Method 1

a support having attached thereto metal ion coordination complexes

Methodology Applied

Scientific EffectMetal ion coordination:

Data Source

PatentUS9874567B2Kits to assay kinase activity

Publication Date: 2018.01.23 STANDARD BIOTOOLS CANADA INC

AI summary

Methods and kits for enzymes involved in post-translational modifications are provided. The methods employ elemental analysis, including ICP-MS. The methods allow for the convenient and accurate analysis of post-translation modifications of substrates by enzymes involved in post-translational modifications, including kinase and phosphatase enzymes. Kits may include an element tag for directly tagging an affinity product that recognizes phosphorylated substrates, as well as a non-phosphorylated substrate or an expression plasmid.