Neutron-encoded mass tags for high-plex proteomics
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current mass spectrometry techniques for proteomic analysis face limitations in achieving high degrees of multiplexing, are susceptible to precursor interference, and are not compatible with a range of dissociation techniques, particularly affecting quantitative accuracy and versatility.
Innovation Solution
The development of methods and systems that utilize isotopic coding agents with small molecular mass differences, combined with high-resolution mass spectrometry, enable accurate quantification of analytes in multiple samples, allowing for high multiplexing capabilities and compatibility with various dissociation techniques like electron capture and electron transfer dissociation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If isobaric tagging methods are used to increase multiplexing capacity, then the number of samples that can be analyzed simultaneously increases, but quantitative accuracy deteriorates due to precursor interference in the MS/MS isolation window
Solution Approach 1:
The patent segments the quantitative information into two distinct stages: MS1 scan for capturing isotopic labels and MS2 scan for peptide identification. This segmentation allows the quantitative data to be extracted from the precursor ion region in MS1, avoiding the precursor interference problem in MS2 that plagues isobaric tagging methods. The isotopic labels are designed to be resolvable in MS1, separating the quantification function from the fragmentation function.
Solution Approach 2:
The patent transitions from the conventional isobaric tagging approach where all tags have the same nominal mass to a neutron encoding scheme where tags have progressively increasing masses (e.g., 0, 1, 2, 3 Da increments). This dimensional change in mass spacing allows resolution of multiple isotopic labels in the MS1 scan while maintaining compatibility with standard collisional activation in MS2, thus achieving both high multiplexing and accurate quantification.
2Measurement precision
If conventional SILAC methods are used with 4 Da spacing to limit isotopic cluster overlap, then measurement precision is maintained, but multiplexing capacity is limited to triplex due to mass spectral complexity
Solution Approach 1:
The patent changes the mass spacing parameter from the conventional 4 Da (SILAC) to progressively smaller increments of 1 Da or less between neutron-encoded isotopic labels. This parameter change is made possible by the use of high-resolution mass spectrometry and neutron-based encoding, which allows resolution of closely spaced isotopic clusters while maintaining quantitative accuracy. The smaller mass differences enable higher multiplexing (e.g., 6-plex, 8-plex, or more) without excessive mass spectral complexity.
Solution Approach 2:
The patent uses multiple isotopic variants (isotopologues) of the same amino acid or peptide sequence as distinct coding agents. Each isotopologue contains a specific number of neutrons (e.g., different combinations of 13C, 15N, 2H, 18O) that create distinct mass differences. This copying approach with neutron-encoded variants allows multiple samples to be distinguished by their precise mass differences while maintaining the same chemical properties for consistent chromatographic and fragmentation behavior.
3Productivity
If isobaric tagging methods are used to achieve high multiplexing, then productivity increases, but adaptability deteriorates because the methods are only compatible with collisional activation for dissociation
Solution Approach 1:
The patent creates a universal labeling system that is compatible with multiple dissociation techniques including collisional activation (CID), electron capture dissociation (ECD), and electron transfer dissociation (ETD). The neutron-encoded isotopic labels are designed to be chemically inert and structurally integrated into the peptide backbone, allowing them to survive various dissociation methods without interfering with fragmentation patterns. This universality enables the same labeling approach to be used across different mass spectrometry platforms and experimental conditions.
4Ease of operation
If isotopic labels with larger mass differences are used to simplify detection, then ease of operation improves, but the degree of multiplexing is reduced due to increased mass spectral complexity
Solution Approach 1:
The patent replaces the mechanical/chemical approach of large mass spacing (conventional SILAC with 4 Da) with a precision-based approach using neutron encoding and high-resolution mass measurement. Instead of relying on large mass differences for easy detection, the system uses precise mass measurement of small differences (1 Da or less) enabled by high-resolution mass spectrometry. This substitution allows higher multiplexing while maintaining detection simplicity through automated peak picking and integration algorithms that can resolve the closely spaced isotopic clusters.
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
These methods provide accurate and high-throughput quantitation of protein abundances, overcoming limitations of precursor interference and expanding the range of applicable dissociation techniques, thereby enhancing the versatility and accuracy of proteomic analysis.
Implementation Method 1
analyzing the isotopically labeled analytes for each sample using a mass spectrometry analysis technique providing a resolving power equal to or greater than 100,000
Implementation Method 2
compatible with a range of dissociation techniques including electron capture and electron transfer dissociation methods
Implementation Method 3
compatible with a range of dissociation techniques including electron capture and electron transfer dissociation methods
Data Source
AI summary
The invention provides mass spectrometry methods, compositions and systems which enable a unique platform for analyte quantitation accessing very high degrees of multiplexing and accurate quantification, particularly well-suited for a range of quantitative analysis for proteomics applications. Embodiments of the present methods and systems combine isotopic coding agents characterized by very small differences in molecular mass with mass spectrometry methods providing large resolving power to provide relative or absolute analyte quantification in a large number of samples.