KRas Peptide Selection for SRM/MRM Quantification in FFPE Tissue
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Solution Overview
Problem
Current methods for detecting KRas protein levels in formalin-fixed cancer tissue samples are inefficient due to unsuitable peptide sequences for mass spectrometry-based SRM/MRM assays, leading to difficulties in ionization, fragmentation, and resolution, making it challenging to accurately quantify the protein for diagnostic and therapeutic purposes.
Innovation Solution
The use of specific KRas fragment peptides, such as SEQ ID NO:1, in a mass spectrometry-based SRM/MRM assay, which involves proteolytic digestion of formalin-fixed tissue using Liquid Tissue protocol and analysis on a triple quadrupole mass spectrometer, allows for relative and absolute quantitation by comparing signature peak areas, including the use of isotopically labeled internal standards for absolute quantitation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional peptide sequences are used in SRM/MRM assays for KRas detection, then the assay can be performed with standard protocols, but the detection accuracy and quantification precision are insufficient due to poor ionization and fragmentation
Solution Approach 1:
The patent applies parameter changes by selecting specific peptide sequences with optimized properties for mass spectrometry detection. The identified peptides (e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3) have specific characteristics including appropriate mass-to-charge ratios, fragmentation patterns, and ionization efficiency that differ from conventional peptides, thereby resolving the contradiction between detectability and measurement precision
Solution Approach 2:
The patent uses synthetic peptide copies as internal standards (isotopically labeled versions of the target peptides) to enable accurate quantification. These synthetic copies replicate the ionization and fragmentation behavior of the endogenous peptides while being distinguishable by mass spectrometry, allowing precise measurement despite the challenges of detecting low-abundance KRas peptides in complex tissue samples
2Reliability
If formalin-fixed tissue is used for KRas analysis, then sample preservation and storage are improved, but protein extraction and peptide detection become more difficult
Solution Approach 1:
The patent applies preliminary action by performing proteolytic digestion of the formalin-fixed tissue to generate the specific KRas peptides before analysis. The Liquid Tissue protocol includes preliminary steps of tissue processing, protein extraction, and enzymatic digestion that convert the fixed tissue into a form suitable for detecting the specific peptides, thereby making the subsequent SRM/MRM analysis feasible despite the challenging fixed tissue matrix
Solution Approach 2:
The patent uses specific proteolytic digestion as an intermediary process that bridges the gap between formalin-fixed tissue and detectable peptides. The digestion step converts the cross-linked proteins in fixed tissue into free peptides that can be extracted and analyzed by mass spectrometry, serving as a necessary intermediate transformation
3Device complexity
If relative quantitation is used in SRM/MRM assays, then the assay complexity is reduced, but absolute quantification capability is lost
Solution Approach 1:
The patent uses synthetic peptide copies (isotopically labeled internal standards) that are identical to the target peptides except for their mass. These copies are added to samples at known concentrations and co-extracted with the endogenous peptides, allowing the instrument to measure the ratio of endogenous to standard peptide signals. This approach enables absolute quantification without significantly increasing assay complexity, as the same SRM/MRM methodology is used for both
Solution Approach 2:
The patent implements feedback through the use of internal standards that provide a reference signal for quantification. The known amount of added synthetic peptide serves as a feedback reference that allows calculation of the absolute amount of endogenous peptide in the sample, correcting for variations in extraction efficiency, ionization, and instrument response
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 enables accurate and precise measurement of KRas protein levels in cancer tissue, providing diagnostic, prognostic, and therapeutic insights, facilitating personalized medicine by correlating protein expression with cancer stage and therapeutic agent selection.
Implementation Method 1
mass spectrometry-based SRM/MRM assay
Implementation Method 2
allows for relative and absolute quantitation by comparing signature peak areas
Implementation Method 3
involves proteolytic digestion of formalin-fixed tissue using Liquid Tissue protocol
Data Source
Figure 1A~1C
AI summary
The current disclosure provides for specific peptides, and derived ionization characteristics of the peptides, from the GTPase KRas Protein (KRas) that are particulariy advantageous for quantifying the KRas 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 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,