SRM/MRM Assay for AXL Protein Quantification in FFPE Tissues
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Solution Overview
Problem
Current cancer therapies, including traditional chemotherapeutic agents and targeted therapies, often fail to differentiate between cancer cells and normal cells, leading to non-specific treatment outcomes, and there is a need for more precise methods to determine the effectiveness of AXL-targeted therapeutic agents based on AXL protein expression levels in cancer patients.
Innovation Solution
A quantitative proteomics-based assay using mass spectrometry, specifically a Selected Reaction Monitoring (SRM)/Multiple Reaction Monitoring (MRM) assay, is developed to measure the activation levels of the AXL signal pathway in formalin-fixed cancer tissues, enabling precise quantification of AXL protein levels for improved treatment decisions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional chemotherapeutic agents are used to kill rapidly dividing cells, then cancer cells are eliminated, but normal growing cells are also damaged
Solution Approach 1:
The invention segments the detection process by using specific peptide sequences from AXL protein as unique markers. Instead of treating all rapidly dividing cells uniformly, the assay identifies and quantifies specific AXL-derived peptides (e.g., from extracellular domain, transmembrane domain, or intracellular domain) to distinguish cancer cells expressing AXL from normal cells, enabling targeted therapy selection without affecting normal cells
2Reliability
If AXL-targeted therapeutic agents are used to treat cancer, then treatment specificity is improved, but only patients with high AXL expression benefit
Solution Approach 1:
The invention implements a feedback mechanism through the SRM/MRM quantitative assay that measures AXL protein levels in patient tumor samples before treatment. This feedback loop allows clinicians to determine whether a patient's cancer cells express sufficient AXL protein to respond to AXL-targeted therapy, thereby adapting treatment decisions to individual patient characteristics and avoiding ineffective treatments for low-AXL patients
3Measurement precision
If mass spectrometry SRM/MRM assay is used to quantify AXL protein, then measurement precision is improved, but assay complexity increases
Solution Approach 1:
The invention extracts and focuses on specific peptide sequences from the AXL protein that serve as unique identifiers and quantitative markers. By selecting particular peptides (e.g., from specific domains of AXL) and using them as targets for SRM/MRM detection, the assay achieves high measurement precision for AXL quantification while managing complexity through targeted rather than comprehensive protein analysis
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
The SRM/MRM assay provides accurate and precise quantitative data on AXL protein levels in cancer tissues, aiding in cancer diagnosis and personalized treatment strategies by correlating protein expression with therapeutic agent effectiveness, thereby enhancing treatment specificity and patient outcomes.
Implementation Method 1
a mass spectrometry assay that quantifies AXL in formalin fixed tissues from cancer patients
Implementation Method 2
The peptide sequence and fragmentation/transition ions for each peptide are particularly useful in a mass spectrometry-based Selected Reaction Monitoring (SRM), which can also be referred to as a Multiple Reaction Monitoring (MRM) assay
Data Source
AI summary
The current disclosure provides for specific peptides, and derived ionization characteristics of the peptides, from the tyrosine-protein kinase receptor UFO protein (AXL) that are particularly advantageous for quantifying the AXL protein directly in biological samples that have been fixed in formalin by the method of Selected Reaction Monitoring (SRM) mass spectrometry, or what can also be termed as Multiple Reaction Monitoring (MRM) mass spectrometry. Such biological samples are chemically preserved and fixed wherein said biological sample is selected from tissues and cells treated with formaldehyde containing agents/fixatives including formalin-fixed tissue/cells, formalin-fixed/paraffin embedded (FFPE) tissue/cells, FFPE tissue blocks and cells from those blocks, and tissue culture cells that have been formalin fixed and or paraffin embedded. A protein sample is prepared from said biological sample using the Liquid Tissue reagents and protocol and the AXL protein is quantitated in the Liquid Tissue sample by the method of SRM/MRM mass spectrometry by quantitating in the protein sample at least one or more of the peptides described. These peptides can be quantitated if they reside in a modified or an unmodified form. An example of a modified form of an AXL peptide is phosphorylation of a tyrosine, threonine, serine, and/or other amino acid residues within the peptide sequence.