Protein Quantification via Normalized SIS Peptide Transitions
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current multiple reaction monitoring (MRM) mass spectrometry techniques face challenges in controlling pre-analytical and analytical variability in blood-based clinical testing, particularly in protein quantification, due to the complexity of the human blood proteome and variations in laboratory conditions.
Innovation Solution
A method involving the generation of peptide transitions from proteins in a biological sample, using both target and normalizing proteins, where the expression level of target proteins is determined by measuring mass spectroscopy signal intensities and calculating response ratios normalized by sample-dependent factors to reduce variability, specifically selecting normalizing proteins to minimize intensity drift and technical coefficient of variation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If stable isotope-labeled internal standard (SIS) peptides are used for protein quantification, then analytical variability in post-digestion procedures is controlled, but pre-analytical variability associated with sample collection and handling cannot be controlled
Solution Approach 1:
The patent applies preliminary action by spiking SIS peptides into samples before protein digestion occurs. This timing allows the internal standards to be present throughout all subsequent analytical procedures, enabling them to control variability in digestion efficiency, peptide extraction, and mass spectrometry analysis. The SIS peptides experience the same pre-analytical and analytical processing steps as the endogenous peptides, thereby compensating for variations introduced at any stage.
2Reliability
If full-length SIS proteins are spiked into samples before analytical procedures, then the best control of analytical variability is achieved, but quality control of production and storage of SIS proteins is challenging
Solution Approach 1:
The patent applies segmentation by breaking down the protein into smaller peptide fragments through enzymatic digestion. Instead of using intact full-length SIS proteins which require stringent quality control for production and storage, the method uses SIS peptides that are more stable and easier to characterize. The digestion process converts the complex protein standard into multiple smaller, more manageable peptide standards that can be individually validated and stored with greater ease while maintaining the ability to control analytical variability.
3Productivity
If MRM-MS is used for fast and reproducible measurement of multiple proteins, then productivity is improved, but controlling analytical variability to satisfy quality control requirements becomes challenging
Solution Approach 1:
The patent applies feedback by using SIS peptides as internal references that provide real-time information about variability introduced during sample processing and analysis. The known amounts of SIS peptides spiked into each sample serve as a feedback mechanism to monitor and correct for variations in digestion efficiency, peptide recovery, and instrument performance. By comparing the measured SIS peptide signals against their expected values, the system can identify and compensate for analytical variability, ensuring quality control requirements are met while maintaining high throughput.
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 robust and simple method to control pre-analytical and analytical variability, improving the accuracy of protein expression level measurements and enhancing the reliability of clinical testing by reducing technical drift and variation, making it suitable for routine clinical testing and biomarker research.
Implementation Method 1
The principle of stable isotope labeling (SIL) is currently used in MS-based quantitative proteomics to control experimental variability. Protein abundance is measured by comparing MS signal intensities of endogenous peptides with those of their corresponding stable isotope-labeled internal standard (SIS) peptides.
Implementation Method 2
A mass spectroscopy (MS) signal intensity is measured from the plurality of respective peptide transitions and a plurality of corresponding stable isotope-labeled internal standard (SIS) peptide transitions.
Data Source
AI summary
Methods are provided for determining the expression level of target proteins in a subject. A plurality of respective peptide transitions are generated from a plurality of proteins obtained from a biological sample from the subject, wherein the plurality of proteins comprises both target and normalizing proteins. A mass spectroscopy (MS) signal intensity is measured from the plurality of respective peptide transitions and a plurality of corresponding stable isotope-labeled internal standard (SIS) peptide transitions. For each of the plurality of proteins, a response ratio is calculated between the MS signal intensity of the respective peptide transition and the corresponding SIS peptide transition. The response ratio for each target protein is normalized by a sample-dependent normalization factor calculated from the response ratio for each normalizing protein, wherein the normalized response ratios provide a determination of the expression level of the target proteins.