Protein Isoform Quantitation via PDITM Mass Spectrometry
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Solution Overview
Problem
Protein expression quantification is challenging due to the lack of correlation between mRNA and protein levels, and proteins are dynamically modified by environmental factors, making traditional methods unreliable for absolute quantitation.
Innovation Solution
The use of parent-daughter ion transition monitoring (PDITM) in mass spectrometry for absolute quantitation of protein isoforms, involving signature peptide-diagnostic daughter ion transitions and normalization proteins to determine protein concentrations in a multiplex fashion.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional protein quantification methods are used, then the process is simpler, but the measurement precision and reliability of absolute quantitation deteriorates
Solution Approach 1:
The method performs preliminary actions by digesting proteins into peptides and synthesizing signature peptide standards before the actual quantification process. These preparatory steps enable the subsequent mass spectrometry analysis to achieve accurate absolute quantitation by having reference standards ready for comparison with sample peptides.
Solution Approach 2:
Signature peptides serve as intermediaries between the target proteins and the detection system. Instead of directly measuring intact proteins, the method uses specific peptide fragments as mediators that can be reliably detected and quantified by mass spectrometry, then uses these measurements to infer protein concentrations.
2Productivity
If multiple proteins are quantified individually, then the measurement precision is maintained, but the productivity and time consumption deteriorates
Solution Approach 1:
The method merges the quantification of multiple proteins into a single multiplexed analysis. By using mass spectrometry to simultaneously detect multiple signature peptides from different proteins in one run, the method achieves high productivity while maintaining measurement precision through the use of specific mass-to-charge ratio detection for each peptide.
Solution Approach 2:
The mass spectrometry system performs multiple functions simultaneously - it can detect and quantify multiple different signature peptides in a single analysis run. This universal detection capability allows the system to handle various proteins with different signature peptides using the same analytical platform and methodology.
3Measurement precision
If signature peptide standards are used for each protein, then the measurement precision improves, but the loss of substance and resource requirements worsen
Solution Approach 1:
The method extracts only the essential signature peptides from the complete protein structures for quantification purposes. Instead of requiring entire proteins or large portions of them as standards, it isolates and uses only the specific peptide fragments that are characteristic and sufficient for identifying and quantifying the parent proteins.
Solution Approach 2:
Synthetic signature peptide standards serve as simplified copies or representations of the actual protein-derived peptides. These synthetic copies can be produced in controlled amounts with known concentrations, providing reliable reference standards without requiring large quantities of the actual biological samples.
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
Enables accurate and reliable absolute quantitation of multiple protein isoforms in a single sample loading, facilitating the assessment of biological states, drug responses, and disease markers.
Implementation Method 1
mass spectrometry system comprises a first mass separator, and ion fragmentor and a second mass separator
Implementation Method 2
transmitted mass-to-charge (m/z) range of a first mass separator
Implementation Method 3
the transmitted m/z range of a first mass separator (often referred to as the first dimension of mass spectrometry) is selected to transmit a molecular ion (often referred to as 'the parent ion' or 'the precursor ion') to an ion fragmentor (e.g., a collision cell, photodissociation region, etc.) to produce fragment ions
Implementation Method 4
loading at least a portion of the combined samples onto a chromatographic column
Data Source
AI summary
In various aspects, the present teachings provide systems, methods, assays and kits for the absolute quantitation of protein expression. In various aspects, the present teachings provide methods of determining the concentration of one or more proteins of interest in one or more samples of interest. In various aspects, the present teachings provide methods of determining the absolute concentration of one or more isoforms of a protein using standard samples of signature protein fragments and parent-daughter ion transition monitoring (PDITM). In various embodiments, the absolute concentration of multiple isoforms of a biomolecule in a sample, multiple proteins in a biological process, a combination of multiple samples, or combinations thereof, can be determined in a multiplex fashion using the present teachings. In various aspects, provided are methods of assessing the response of a biological system to a chemical agent.