SRM/MRM Assay for DR5 Protein Quantification in FFPE Tissues
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Solution Overview
Problem
Current methods are inadequate for accurately measuring the DR5 protein levels in formalin-fixed tissue samples using mass spectrometry due to challenges in peptide detection and modification analysis, leading to unreliable results.
Innovation Solution
A method employing specific DR5 fragment peptides and mass spectrometry-based Selected Reaction Monitoring (SRM/MRM) assays, involving peptide digestion, fractionation, and isotopically labeled internal standards, to quantify DR5 protein levels in formalin-fixed tissue samples, enabling both relative and absolute quantification and correlation with cancer diagnosis and therapy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If mass spectrometry is used to measure DR5 protein levels in formalin-fixed tissue, then protein quantification is achieved, but detection accuracy is insufficient due to peptide modification analysis challenges
Solution Approach 1:
The patent segments the complex DR5 protein measurement problem into specific peptide detection tasks. By identifying and targeting particular peptide sequences (e.g., peptides containing tryptophan residues) within the DR5 protein, the method breaks down the difficult whole-protein measurement into more manageable and detectable peptide-level analyses, thereby improving measurement precision while addressing detection difficulties
Solution Approach 2:
The patent introduces isotopically labeled internal standards as intermediaries to facilitate accurate DR5 protein quantification. These labeled peptides serve as reference points that co-elute with endogenous peptides, enabling precise relative quantification by comparing signal intensities between labeled and unlabeled peptides, thus overcoming the detection accuracy limitations
2Stability of the object's composition
If formalin-fixed tissue is used for analysis, then tissue preservation is achieved, but protein extraction and peptide detection become more challenging
Solution Approach 1:
The patent applies preliminary action by performing specific protease digestion (e.g., trypsin digestion) on the formalin-fixed tissue before mass spectrometry analysis. This pre-digestion step converts the fixed tissue proteins into peptides that are more amenable to extraction and detection, effectively bridging the gap between tissue preservation and analytical accessibility
Solution Approach 2:
The patent employs parameter changes by optimizing digestion conditions (enzyme type, temperature, time) and mass spectrometry parameters (collision energy, precursor ion selection) to specifically enhance the detection of DR5-derived peptides from formalin-fixed tissue, thereby making the analysis feasible despite the fixed tissue state
3Measurement precision
If specific DR5 fragment peptides are targeted for SRM/MRM analysis, then quantification precision is improved, but assay complexity increases
Solution Approach 1:
The patent achieves universality by designing an SRM/MRM assay that can simultaneously monitor multiple DR5 peptide sequences in a single analysis run. By selecting peptides with characteristic features (e.g., tryptophan residues that produce diagnostic ions), the method creates a multi-functional assay that provides comprehensive DR5 quantification information while maintaining practical simplicity through standardized protocols
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 precise and accurate measurement of DR5 protein levels, aiding in cancer diagnosis and personalized treatment decisions by correlating protein expression with cancer stage and therapeutic responses.
Implementation Method 1
detecting and/or quantifying the level of a DR5 fragment peptide in a protease digest of said biological sample using mass spectrometry
Implementation Method 2
a protease digest of said biological sample
Implementation Method 3
fractionating said protease digest prior to detecting and/or quantifying the level of said DR5 fragment peptide, wherein said fractionating step is selected from the group consisting of liquid chromatography
Data Source
AI summary
Specific peptides, and derived ionization characteristics of those peptides from Death Receptor 5 (DR5) protein are provided that are particularly advantageous for quantifying the DR5 protein directly in biological samples that have been fixed in formalin by the method of Selected Reaction Monitoring / Multiple Reaction Monitoring (SRM/MRM) mass spectrometry. Such biological samples are chemically preserved and fixed wherein 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 a biological sample using the Liquid Tissue™ reagents and protocol, and the DR5 protein are 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 for one or more of the DR5 protein. These peptides can be quantitated if they reside in a modified or in an unmodified form. An example of a modified form of a DR5 peptide is phosphorylation of a tyrosine, threonine, serine, and/or other amino acid residues within the peptide sequence