Mass Spectrometry Biomolecule Quantitation Using Stable Isotope Standards
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Solution Overview
Problem
Current diagnostic methods for neurological and neurodegenerative diseases, such as Alzheimer's, lack sensitivity and accuracy in quantifying diagnostic biomolecules in blood and cerebral spinal fluid before the onset of clinical symptoms, and cannot effectively measure the effects of treatments to prevent or slow disease progression.
Innovation Solution
A method using a Quantitation Internal Standard, such as a stable isotope labeled biomolecule, to calculate the concentration of endogenous biomolecules in samples by determining the ratio of labeled to unlabeled biomolecules, allowing for precise measurement of proteins, peptide fragments, and proteoforms in plasma, serum, or CSF, and monitoring changes induced by therapeutic interventions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If ELISA assays are used to measure biomolecule concentrations, then antibody-based detection can be performed, but high inter- and intra-assay variability and limited multiplexing capability result
Solution Approach 1:
The patent replaces the mechanical/chemical ELISA assay system with a mass spectrometry-based system. Instead of using antibodies that bind to proteins and require colorimetric or fluorescent detection, the invention uses mass spectrometry to directly measure the mass-to-charge ratio of peptide fragments derived from biomolecules. This substitution eliminates the variability associated with antibody binding and enables simultaneous measurement of multiple biomolecules in a single assay.
2Measurement precision
If antibodies are used for specific protein detection, then isoform-specific quantitation can be achieved, but reliance on two antibodies binding can lead to high variability in reported concentrations
Solution Approach 1:
The patent extracts the detection step from the complex two-antibody ELISA system and replaces it with a single mass spectrometry detection step. By digesting proteins into peptide fragments and directly analyzing these fragments by mass spectrometry, the invention eliminates the need for capture and detection antibodies, thereby removing the source of variability while simplifying the overall assay procedure.
Solution Approach 2:
The patent introduces peptide fragments as intermediaries between the original protein and the detection system. Instead of directly detecting intact proteins with antibodies, the proteins are first digested into characteristic peptide fragments that serve as unique identifiers and quantification targets for mass spectrometry, enabling more precise and reproducible measurements.
3Loss of time
If current diagnostic methods are used, then clinical diagnosis can be made, but extensive neuronal loss has already occurred by the time of diagnosis
Solution Approach 1:
The patent replaces conventional ELISA-based diagnostic methods with mass spectrometry, which provides significantly higher sensitivity and precision for biomolecule quantification. This enhanced measurement capability allows for the detection of biomolecular changes at earlier stages of disease progression, before extensive neuronal loss occurs and before clinical symptoms manifest.
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 sensitive, accurate, and reproducible quantification of biomolecules, facilitating early diagnosis and effective treatment monitoring, optimizing therapeutic interventions and predicting individual responses to specific agents based on biomolecule concentrations.
Implementation Method 1
contacting a sample from the subject with a Quantitation Internal Standard, where the Quantitation Internal Standard is a known concentration of a labeled biomolecule of interest
Implementation Method 2
determining a ratio of labeled Quantitation Internal Standard to unlabeled endogenous biomolecules in the sample
Data Source
AI summary
The present invention provides methods for measuring the absolute concentration of Tau, and other protein, peptide fragments and proteoforms in CSF and plasma samples collected from a subject. Such biomolecules may be implicated in one or more neurological and neurodegenerative diseases or disorders. Also provided is a method for determining whether a therapeutic agent affects the CSF or plasma concentration of a central nervous system derived biomolecule. Also provided are kits for performing the methods of the invention.