Her2 SRM Assay Peptides for FFPE Tissue Analysis
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Solution Overview
Problem
Existing mass spectrometry-based SRM/MRM assays face challenges in detecting and quantifying Her2 protein peptides from formalin fixed, paraffin embedded tissue due to peptide instability and inefficiency, limiting their use in determining full-length versus truncated forms of the Her2 protein for cancer diagnosis and treatment.
Innovation Solution
Specific peptides derived from the Her2 protein, optimized for mass spectrometry analysis, are used in an SRM/MRM assay to detect and quantify both full-length and truncated forms of the Her2 protein in formalin fixed tissue samples, employing a combination of intracellular and extracellular peptides and isotopically labeled internal standards for accurate measurement.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If existing mass spectrometry-based SRM/MRM assays are used to detect Her2 protein peptides from formalin fixed tissue, then the assay can be performed on archived tissue samples, but the detection precision and quantification accuracy are limited due to peptide instability and inefficiency
Solution Approach 1:
The patent applies preliminary action by performing proteolytic digestion of the Her2 protein into specific peptides before mass spectrometry analysis. This pre-processing step converts the unstable full-length protein into more stable peptide fragments that are better suited for SRM/MRM detection, thereby improving measurement precision while maintaining reliability on archived formalin-fixed tissue samples.
Solution Approach 2:
The patent employs parameter changes by optimizing peptide selection based on specific mass-to-charge ratios and fragmentation patterns. By carefully selecting peptides with appropriate stability characteristics and detection parameters, the assay achieves both high measurement precision and reliability despite the challenges of analyzing formalin-fixed tissue samples.
2Difficulty of detecting and measuring
If existing SRM/MRM assays are used, then the assay structure is established, but the ability to distinguish full-length versus truncated forms of Her2 protein is insufficient
Solution Approach 1:
The patent applies segmentation by selecting multiple specific peptides from different regions of the Her2 protein sequence, including both full-length and truncated forms. This segmentation allows the mass spectrometry assay to detect and distinguish between different protein forms by monitoring specific peptide signatures, thereby improving detection capability while preserving protein form information.
Solution Approach 2:
The patent uses isotopically labeled internal standard peptides as intermediaries to enhance the detection and quantification of endogenous Her2 peptides. These labeled standards serve as reference points that improve the accuracy of distinguishing between full-length and truncated forms by providing known markers for comparison.
3Quantity of substance
If formalin fixed tissue samples are used, then archived patient tissue can be analyzed, but peptide recovery and detection efficiency are reduced
Solution Approach 1:
The patent applies preliminary action by implementing optimized tissue extraction and proteolytic digestion protocols specifically designed for formalin-fixed paraffin-embedded (FFPE) tissues. These pre-processing steps enhance peptide recovery from archived samples by improving antigen retrieval and protease accessibility, thereby increasing the quantity of detectable peptides without compromising sample processing efficiency.
Solution Approach 2:
The patent employs parameter changes by optimizing digestion conditions, including protease selection, incubation temperature, and time parameters, to maximize peptide recovery from formalin-fixed tissue. These parameter optimizations improve the efficiency of converting fixed tissue into detectable peptide forms while maintaining practical sample processing workflows.
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 method enables precise detection and quantification of Her2 protein forms in cancer tissues, providing critical diagnostic and therapeutic insights for personalized cancer treatment by distinguishing between full-length and truncated forms, thereby predicting drug resistance and selecting appropriate therapeutic agents.
Implementation Method 1
detecting specific peptides from the Her2 protein in a protein digest prepared from said biological sample using mass spectrometry
Data Source
AI summary
This disclosure provides ten (10) specific peptides, and particular peptide characteristics, from the cell membrane-bound Her2 protein and a diagnostic assay useful for determining the presence and amount of full length and truncated versions of the full-length Her2 protein in cells derived from formalin fixed paraffin embedded tissue.