SRM/MRM Assay for BRAF Protein Quantification in FFPE Tissue
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Solution Overview
Problem
Current methods for analyzing BRAF protein levels in formalin-fixed cancer tissue samples are inefficient due to the unsuitability of many peptide sequences for mass spectrometry-based SRM/MRM assays, leading to difficulties in detecting and quantifying the protein accurately.
Innovation Solution
The use of specific BRAF fragment peptides, such as SEQ ID NO:4, 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 of BRAF protein levels by comparing signature peak areas, with the option of using an isotope-labeled internal standard for absolute quantitation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If mass spectrometry-based SRM/MRM assays are used to analyze BRAF protein levels in formalin-fixed cancer tissue samples, then quantitative data can be obtained, but many peptide sequences are unsuitable for detection leading to inaccurate quantification
Solution Approach 1:
The patent applies parameter changes by selecting specific peptide sequences with optimized properties for mass spectrometry detection. The invention identifies and uses particular BRAF-derived peptides (such as those with specific amino acid compositions and fragmentation patterns) that exhibit superior ionization efficiency and detectability in SRM/MRM assays, thereby resolving the detection difficulty while maintaining measurement precision
2Quantity of substance
If formalin-fixed tissue is used for BRAF analysis, then tissue preservation and availability are improved, but protein extraction and peptide detection become more difficult
Solution Approach 1:
The patent applies preliminary action by pre-optimizing the selection of BRAF peptide sequences before formalin fixation is performed. The identified peptides are chosen specifically because they remain detectable and quantifiable after formalin fixation and paraffin embedding processes. This preliminary selection ensures that when tissue is fixed and stored, the target peptides remain suitable for later SRM/MRM analysis, thereby easing the subsequent protein extraction and detection steps
3Productivity
If relative quantitation is performed by comparing signature peak areas, then quantification can be achieved, but absolute quantitation requires additional internal standards increasing assay complexity
Solution Approach 1:
The patent applies partial action by implementing a tiered quantification approach. The assay is designed to provide relative quantitation through direct comparison of signature peak areas, which can be achieved quickly and efficiently. The option for absolute quantitation using isotope-labeled internal standards is available but not required for all applications. This partial implementation allows users to obtain sufficient quantitative data for their needs without always incurring the additional complexity and cost of internal standards
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 method provides accurate and precise quantitative data for BRAF protein levels, enabling diagnostic, prognostic, and therapeutic insights, aiding in cancer staging and treatment decisions by correlating protein expression with therapeutic agents, thus facilitating personalized medicine approaches.
Implementation Method 1
analysis on a triple quadrupole mass spectrometer
Implementation Method 2
proteolytic digestion of formalin-fixed tissue using Liquid Tissue protocol
Implementation Method 3
using an isotope-labeled internal standard for absolute quantitation
Data Source
Figure 1A~1C
AI summary
The current disclosure provides for specific peptides, and derived ionization characteristics of the peptides, from the Serine/Threoninc-Protein Kinase B-raf (BRAF) that are particularly advantageous for quantifying the BRAF 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 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.