MRM-MS Detection of Circulating Histones H3 and H2B
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Solution Overview
Problem
Current methods for detecting circulating histones in plasma for sepsis diagnosis and prognosis are plagued by low sensitivity and specificity, leading to unreliable results and poor reproducibility, which hinders early identification and effective treatment of sepsis and septic shock.
Innovation Solution
The use of targeted mass spectrometry-based methods, specifically multiple reaction monitoring (MRM-MS), with heavily labeled Spike-In peptides for the detection and quantification of circulating histones H3 and H2B, providing a more sensitive and specific approach for monitoring sepsis and septic shock.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If immunoassays are used for detection of free histone proteins circulating in plasma, then the method is simple to operate, but the sensitivity and specificity are low leading to unreliable results
Solution Approach 1:
The patent replaces the mechanical/chemical immunoassay system with a mass spectrometry-based detection system. This substitution eliminates the limitations of antibody-based methods (cross-reactivity, autoantibodies, variability between kits) while achieving superior sensitivity and specificity for histone detection in plasma samples.
Solution Approach 2:
The patent changes the detection parameters by using targeted mass spectrometry with multiple reaction monitoring (MRM) and heavy isotope-labeled internal standards. This allows precise quantification of histone peptides at very low concentrations, achieving sensitivity and specificity that immunoassays cannot provide.
2Adaptability or versatility
If immunoassays with different proprietary antibodies are used, then various kits are available for detection, but there is poor concordance between assays and low reproducibility
Solution Approach 1:
The patent establishes a universal mass spectrometry-based platform that can detect multiple histone types (H2A, H2B, H3, H4) and their modifications using a single standardized methodology. This eliminates the variability between different commercial immunoassay kits while maintaining the ability to detect diverse histone species.
Solution Approach 2:
The patent introduces heavy isotope-labeled histone peptides as internal standards that serve as intermediaries for quantification. These labeled standards co-elute with endogenous peptides and provide a reference for accurate measurement, ensuring reproducibility across different laboratories and experiments.
3Ease of manufacture
If immunoassays are used for histone detection, then the method can be performed with available kits, but anti-reagent antibodies and endogenous autoantibodies cause interferences and erroneous results
Solution Approach 1:
The patent replaces the antibody-based detection mechanism with mass spectrometry, which directly detects and quantifies histone peptides based on their mass-to-charge ratio. This eliminates all interference from anti-reagent antibodies and endogenous autoantibodies that plague immunoassays, providing reliable and interference-free measurements.
Data Source
AI summary
The present invention proposes a novel method for the detection of circulating histones H3 and H2B in plasma from sepsis or septic shock (SS) patients, based on mass spectrometry-based methods, in particular based on multiple reaction monitoring targeted mass spectrometry (MRM-MS). Such methods allow quantification of histones using an internal standard and show strong specificity and sensitivity values.