cMET Peptide SRM Assay for FFPE Tissue Quantification
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Solution Overview
Problem
Current methods for analyzing the cMET protein in formalin-fixed cancer tissue samples are inefficient due to the difficulty in detecting specific peptides using mass spectrometry, as many tryptic peptides from the cMet protein do not ionize well or produce distinct fragments, making it challenging to develop reliable and accurate SRM/MRM assays.
Innovation Solution
The use of specific peptides derived from the cMET protein, identified through experimental determination in Liquid Tissue lysates, for mass spectrometry-based SRM/MRM assays, which involve protease digestion, fractionation techniques like nano-reversed phase liquid chromatography, and isotopically labeled internal standards to quantify relative and absolute levels of cMET protein in biological samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If tryptic peptides from cMET protein are used for mass spectrometry analysis, then the protein can be digested into fragments, but the peptides do not ionize well or produce distinct fragments making detection unreliable
Solution Approach 1:
The patent segments the cMET protein into specific tryptic peptides with known sequences and characteristics. By identifying and selecting particular peptide segments (e.g., peptides with specific amino acid compositions that ionize well), the method enables reliable detection of the parent protein through its fragments in mass spectrometry-based SRM/MRM assays.
Solution Approach 2:
The patent changes the parameters of the peptides used for analysis by selecting specific tryptic peptides from cMET that have optimal ionization properties and produce distinct fragmentation patterns. This parameter selection (m/z ratios, fragmentation efficiency) resolves the detection difficulty while maintaining reliability.
2Duration of action of stationary object
If formalin fixation is used to preserve cancer tissue samples, then the tissue can be stored long-term, but the protein analysis becomes more challenging
Solution Approach 1:
The patent performs preliminary identification and characterization of tryptic peptides from cMET in formalin-fixed tissue before developing the SRM/MRM assay. By pre-determining which peptides are present and their ionization characteristics in fixed tissue, the method overcomes the detection challenges posed by formalin fixation while maintaining long-term storage capability.
3Measurement precision
If SRM/MRM assay is developed for cMET protein quantification, then accurate measurement can be achieved, but the assay development becomes complex due to peptide selection challenges
Solution Approach 1:
The patent extracts and identifies specific tryptic peptides from the complex cMET protein that are suitable for SRM/MRM analysis. By selecting particular peptides with optimal properties (ionization efficiency, distinct fragmentation), the method simplifies the assay development process while maintaining measurement precision.
Solution Approach 2:
The patent uses specific tryptic peptides as intermediaries between the parent cMET protein and the mass spectrometry detection system. These peptide intermediaries have optimized properties for ionization and fragmentation, serving as reliable surrogates for quantifying the parent protein while reducing assay complexity.
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 accurate and precise measurement of cMET protein levels in formalin-fixed tissue samples, facilitating cancer diagnosis and personalized treatment decisions by correlating protein expression with therapeutic agents, thereby improving diagnostic and therapeutic outcomes.
Implementation Method 1
detecting and/or quantifying the amount of one or more modified or unmodified cMET fragment peptides in a protein digest prepared from said biological sample using mass spectrometry
Implementation Method 2
fractionation techniques like nano-reversed phase liquid chromatography
Data Source
AI summary
Specific peptides are provided, and derived ionization characteristics of those peptides, from the Hepatocyte Growth Factor Receptor (cMET) protein. The peptides are particularly and surprisingly advantageous for quantifying by the method of Selected Reaction Monitoring (SRM) mass spectrometry the cMET protein directly in biological samples that have been fixed in formalin, or what can also be termed as Multiple Reaction Monitoring (MRM) mass spectrometry. Such biological samples are chemically preserved and fixed where the 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 the biological sample using the Liquid Tissue™ reagents and protocol and the cMET 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 a cMET peptide is phosphorylation of a tyrosine, threonine, serine, and/or other amino acid residues within the peptide sequence.