Isotopically-Labeled Proteome Standards for Mass Spectrometry Quantitation
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Solution Overview
Problem
Current mass spectrometry methods for protein quantitation are limited by unreliable comparative quantification due to varying ionization efficiencies and the inability to accurately compare proteins, especially for low-abundance and membrane proteins, and they lack the capability for absolute quantitation and application to non-cultured tissue samples.
Innovation Solution
The development of isotopically-labeled biomolecule standards, synthesized using recombinant methods, which can be used as internal standards in mass spectrometry to enable accurate absolute and relative quantitation of proteins in samples, including those from non-cultured tissues, by introducing a known quantity of isotopically-labeled biomolecules that behave similarly to the proteins of interest.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional mass spectrometry methods are used for protein quantitation, then protein identification can be achieved, but accurate quantification is unreliable due to varying ionization efficiencies
Solution Approach 1:
The patent changes the chemical composition parameter by introducing isotopically-labeled standard proteins with known concentrations into the sample. These standards have identical ionization characteristics to endogenous proteins but distinguishable mass spectra, allowing reliable quantification by comparing peak intensities between labeled standards and unlabeled endogenous proteins, thereby eliminating the reliability issue caused by varying ionization efficiencies
Solution Approach 2:
The patent uses isotopically-labeled standard proteins as intermediary reference compounds. These intermediaries serve as bridge elements that connect the unknown endogenous proteins to quantifiable measurements, enabling accurate determination of protein concentrations through comparative analysis of mass spectral peak intensities
2Quantity of substance
If 2D gel electrophoresis is used for protein separation and quantitation, then visual separation of proteins can be achieved, but low-abundance and membrane proteins are underrepresented or undetectable
Solution Approach 1:
The patent extracts the limitation of 2D gel electrophoresis by removing the requirement for visual separation and spot intensity measurement. Instead, it uses mass spectrometry with isotopically-labeled standards to directly quantify proteins in solution, thereby extracting and detecting low-abundance and membrane proteins that are otherwise lost or underrepresented in gel-based methods
Solution Approach 2:
The patent substitutes the mechanical separation system of 2D gel electrophoresis with a mass spectrometry-based analytical system. This replacement eliminates the physical limitations of gel separation that cause underrepresentation of certain protein classes, enabling detection and quantification of low-abundance and membrane proteins through their mass spectral signatures
3Measurement precision
If isotopically-labeled standards are introduced for quantitation, then absolute quantitation becomes possible, but the complexity of the analysis increases
Solution Approach 1:
The patent applies preliminary action by pre-synthesizing isotopically-labeled standard proteins with known concentrations before the actual quantitation experiment. These standards are prepared in advance and added to samples as internal references, enabling absolute quantitation without increasing the complexity of the core mass spectrometry analysis procedure
Solution Approach 2:
The patent uses copying by creating isotopically-labeled copies of endogenous proteins that have identical chemical and physical properties but distinguishable mass. These copied standards behave identically to the target proteins during sample preparation and ionization, allowing accurate quantitation while maintaining analytical simplicity through direct peak intensity comparison
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 quantitation of proteins, including low-abundance and membrane proteins, and extends the application to non-cultured samples, providing both relative and absolute concentration information, thereby overcoming the limitations of existing methods.
Implementation Method 1
mass spectrometry (MS) is a powerful analytical technique that is used to identify unknown compounds, to relatively quantify known compounds, and to elucidate the structure and chemical properties of molecules
Data Source
AI summary
The invention provides methods for quantifying biomolecules, such as polypeptides in mass spectrometric analysis. The methods include use of a biomolecule standard having at least one atomic isotope different than that of the naturally occurring isotopes in the biomolecule of interest. Methods of the present invention also include methods for quantifying biomolecules where the copy biomolecule standard is made by expressing the biomolecule using a recombinant cell. Further included are the biomolecule standards themselves, method for making such standards, kits, systems, reagents, and engineered cells relating to the use of biomolecule standards in mass spectrometric analysis.