SRM MRM Assay Peptide Selection for Cancer Protein Quantitation
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Solution Overview
Problem
Current mass spectrometry-based SRM/MRM assays face challenges in accurately measuring specific peptides from estrogen receptor (ER), progesterone receptor (PR), and Ki67 proteins due to issues like poor ionization, peptide adherence, and lack of distinct fragmentation, making it difficult to develop reliable assays for cancer diagnosis and therapy determination.
Innovation Solution
The development of specific peptide sequences and fragmentation/transition ions for SRM/MRM assays, which involve proteolytic digestion of formalin-fixed tissue using Liquid Tissue™ protocol, followed by mass spectrometry analysis to identify suitable peptides for quantitative analysis of ER, PR, and Ki67 proteins, enabling relative and absolute quantitation of these proteins in cancer tissue samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional mass spectrometry-based SRM/MRM assays are used to measure peptides from ER, PR, and Ki67 proteins, then quantitative analysis can be performed, but poor ionization, peptide adherence, and lack of distinct fragmentation occur making accurate measurement difficult
Solution Approach 1:
The patent applies parameter changes by optimizing multiple assay parameters including selecting specific peptide sequences with optimal properties, adjusting mass spectrometry instrumentation parameters, modifying sample preparation conditions, and tuning fragmentation parameters to achieve distinct fragmentation patterns and improved ionization efficiency for reliable quantitative measurement
Solution Approach 2:
The patent replaces conventional mechanical/sample preparation methods with a specialized Liquid Tissue protocol that uses automated tissue processing and proteolytic digestion under controlled conditions, substituting manual procedures with a standardized system that reduces variability and improves measurement reliability
2Measurement precision
If specific peptide sequences are developed for SRM/MRM assays, then quantitative analysis of ER, PR, and Ki67 proteins can be achieved, but the assay development becomes complex requiring optimization of multiple parameters
Solution Approach 1:
The patent applies segmentation by dividing the complex assay development into distinct modules: tissue processing, proteolytic digestion, peptide selection, mass spectrometry analysis, and data interpretation. Each module is optimized independently with specific peptide sequences selected for each target protein (ER, PR, Ki67), allowing systematic development and validation of the complete quantitative assay
Solution Approach 2:
The patent creates a universal Liquid Tissue protocol and standardized peptide selection criteria that can be applied across multiple tissue types and target proteins. The same general approach and methodology are used for quantifying ER, PR, and Ki67 proteins, as well as other cancer biomarkers, reducing overall assay development complexity through method standardization
3Adaptability or versatility
If formalin-fixed tissue is used for analysis, then diagnostic samples from cancer patients can be analyzed, but peptide extraction and ionization efficiency are reduced
Solution Approach 1:
The patent applies preliminary action by performing automated tissue processing and proteolytic digestion on formalin-fixed tissue before mass spectrometry analysis. The Liquid Tissue protocol includes pre-processing steps that reverse formalin cross-linking and extract peptides under optimized conditions, preparing the sample in advance to overcome the negative effects of formalin fixation on subsequent analysis
Solution Approach 2:
The patent introduces an intermediary processing step using the Liquid Tissue protocol as a mediator between formalin-fixed tissue and mass spectrometry analysis. This intermediary system includes specific buffers, enzymes, and processing conditions that facilitate peptide extraction from fixed tissue, bridging the gap between diagnostic sample availability and analytical requirements
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 allows for precise measurement of protein levels in cancer tissues, aiding in cancer diagnosis and personalized medicine by correlating protein expression with cancer stage and therapeutic response, providing diagnostic and therapeutic insights for breast cancer and other cancers.
Implementation Method 1
mass spectrometry-based Selected Reaction Monitoring (SRM), which also can be referred to as a Multiple Reaction Monitoring (MRM) assay
Implementation Method 2
Results from the SRM/MRM assays can be used to measure relative or absolute quantitative levels of one or more of the specific peptides from (ER), the progesterone receptor (PR), and the antigen Ki67 (Ki67) proteins
Implementation Method 3
proteolytic digestion of formalin-fixed tissue using Liquid Tissue protocol
Data Source
AI summary
The present invention is related to a method for measuring the amount of the human Progesterone Receptor (PR) protein in a biological sample of formalin-fixed tissue, comprising detecting and quantifying the amount of an PR fragment peptide in a protein digest prepared from said biological sample using mass spectrometry; and calculating the amount of PR protein in said sample; wherein said PR fragment peptide is selected from the group consisting of a PR fragment peptide of SEQ ID NO: 4 and a PR fragment peptide of SEQ ID NO: 5; and wherein the amount is a relative amount or an absolute amount.