SRM/MRM Assay for EPHA2 Protein Quantification in FFPE Tissue
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Solution Overview
Problem
Current methods for analyzing the Ephrin Type-A Receptor 2 protein in formalin-fixed cancer tissue samples using mass spectrometry-based SRM/MRM assays face challenges in identifying suitable peptides due to ionization issues and peptide modifications, leading to inaccurate results.
Innovation Solution
Specific peptides derived from the Ephrin Type-A Receptor 2 protein, such as those listed in Table 1, are identified and used in SRM/MRM assays, along with isotopically labeled internal standards, to quantify the protein levels accurately in formalin-fixed tissue samples, employing mass spectrometry and proteolytic digestion protocols like Liquid Tissue™, to overcome detection difficulties and modifications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional mass spectrometry-based SRM/MRM assays are used to analyze EPHA2 protein in formalin-fixed cancer tissue, then the assay can detect protein levels, but ionization issues and peptide modifications lead to inaccurate results
Solution Approach 1:
The patent applies parameter changes by systematically varying peptide selection criteria to identify sequences that resist formalin-induced modifications and ionization suppression. Specific peptides with particular amino acid compositions and sequences were selected based on their resistance to carbamylation and other formalin-related modifications, thereby changing the detection parameters to achieve accurate quantification in formalin-fixed tissues.
Solution Approach 2:
The patent uses stable isotope-labeled internal standards as intermediaries to compensate for ionization issues and peptide modifications. These labeled peptides serve as reference points that co-elute with native peptides but can be distinguished by mass spectrometry, allowing accurate quantification despite modifications or ionization suppression in the formalin-fixed tissue samples.
2Reliability
If formalin fixation is used to preserve cancer tissue samples, then tissue preservation is achieved, but peptide modifications occur that interfere with mass spectrometry detection
Solution Approach 1:
The patent converts the harmful effect of formalin-induced peptide modifications into a beneficial selection criterion. By identifying which peptides remain stable and detectable despite formalin fixation, the method transforms the preservation process's negative impact into a useful filter for selecting robust analytical targets that can withstand routine clinical tissue preparation.
Solution Approach 2:
The patent changes the peptide selection parameters to specifically target sequences that are resistant to formalin-induced carbamylation and other modifications. This involves selecting peptides with specific amino acid compositions that are less susceptible to formalin reactions, thereby maintaining tissue preservation benefits while minimizing detection interference.
3Loss of information
If complex protein lysate samples from patient tissue are analyzed, then comprehensive protein information is obtained, but detection accuracy decreases due to sample complexity
Solution Approach 1:
The patent extracts specific informative peptides from the complex protein lysate that are most suitable for SRM/MRM analysis. By focusing on a selected subset of peptides with known sequences and resistance to modifications, the method extracts the essential quantitative information needed for accurate EPHA2 measurement while avoiding the interference of other complex matrix components.
Solution Approach 2:
The patent segments the complex protein analysis into targeted peptide detection. Instead of attempting to analyze all proteins in the lysate simultaneously, the method divides the problem into detecting specific EPHA2-derived peptides with known characteristics, thereby achieving accurate quantification of the target protein while managing sample 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
The described method enables precise relative and absolute quantification of EPHA2 protein levels in cancer tissue, aiding in cancer diagnosis and therapeutic agent selection, providing a personalized medicine approach by accurately measuring protein expression levels in complex samples.
Implementation Method 1
proteolytic digestion protocols like Liquid Tissue™
Implementation Method 2
mass spectrometry-based Selected Reaction Monitoring (SRM) assay
Implementation Method 3
measuring the amount of the EPHA2 protein in a given protein preparation obtained from a biological sample by mass spectrometry
Implementation Method 4
isotopically labeled internal standards, to quantify the protein levels accurately
Data Source
AI summary
Specific peptides, and derived ionization characteristics of the peptides, from the Ephrin Type-A Receptor 2 (EPHA2) protein are provided that are particularly advantageous for quantifying the EPHA2 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 and are 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 EPHA2 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 EPHA2 peptide is phosphorylation of a tyrosine, threonine, serine, and/or other amino acid residues within the peptide sequence.