Bifunctional Linker Mass Labeling for Phosphopeptide Analysis
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Solution Overview
Problem
Current methods for labeling biomolecules for mass spectrometry analysis, particularly for multiplexing and post-translational modification analysis, face challenges in ease of synthesis and efficiency, especially in handling phosphopeptides and glycopeptides, which often result in reduced sensitivity and incomplete reactions due to the presence of phosphate groups and complex sample handling.
Innovation Solution
A method involving bifunctional linkers and mass labeling reagents for two-step reactions on solid phase supports, allowing for easier synthesis and high-level multiplexing, with pH-controlled coupling reactions and organic solvent elution for cleaner mass spectra, and specific enrichment and depletion steps for phosphate and saccharide groups, enhancing the detection and quantification of phosphorylated and glycosylated peptides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple isobaric mass tag sets with offset masses are used to increase multiplexing rates, then the overall plexing rate is improved, but the size and complexity of individual tags increases
Solution Approach 1:
The invention divides the mass tag system into separate components: a core isobaric mass tag set and multiple mass series modifiers. Each modifier can be independently combined with the core tags to create different offset mass sets, allowing high multiplexing capability without increasing the size of individual tag molecules.
Solution Approach 2:
The core isobaric mass tag set serves as a universal platform that can be combined with multiple different mass series modifiers to generate multiple offset mass sets. This multi-functional approach allows a single core tag structure to support multiple multiplexing configurations without requiring separate tag syntheses for each offset.
2Adaptability or versatility
If multiple mass tag sets are synthesized individually to achieve high multiplexing, then the multiplexing capability is improved, but the synthesis time and cost increases
Solution Approach 1:
The invention performs preliminary synthesis of a single core isobaric mass tag set and pre-preparation of multiple mass series modifiers. These pre-synthesized components can then be easily combined in subsequent steps to create multiple offset mass sets, eliminating the need to synthesize complete tag sets from scratch for each multiplex configuration.
Solution Approach 2:
By segmenting the tag system into a core set and modular modifiers, the invention enables independent synthesis and storage of each component. This segmentation allows the components to be pre-synthesized and then rapidly assembled into different multiplex configurations without repeating the entire synthesis process.
3Ease of operation
If phosphopeptides are analyzed directly without enrichment, then the sample handling is simplified, but the detection sensitivity decreases due to phosphate group interference
Solution Approach 1:
The invention extracts and removes phosphate groups from phosphopeptides through beta-elimination reactions before mass spectrometry analysis. This extraction of the interfering phosphate moiety eliminates the interference while preserving the peptide backbone for accurate mass spectrometric detection, thereby improving sensitivity without significantly complicating the overall sample handling workflow.
4Adaptability or versatility
If complex multi-step labelling protocols are used for post-translational modification analysis, then the analysis capability is improved, but the reaction efficiency decreases
Solution Approach 1:
The invention extracts phosphate groups from phosphopeptides through beta-elimination, creating reactive intermediates that can be directly labeled in a single step with mass tag reagents. This extraction step simplifies the overall protocol by eliminating the need for multiple separate labeling steps, thereby improving reaction efficiency while maintaining comprehensive analysis capability for post-translational modifications.
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 simplifies the synthesis of mass labels, improves multiplex analysis efficiency, and enhances the detection and quantification of post-translational modifications, particularly for phosphopeptides and glycopeptides, by reducing over-labeling and improving reaction completeness, leading to cleaner mass spectra and better sample handling.
Implementation Method 1
contacting the mixture of captured peptides on the hydrophobic solid support with one or more bifunctional linker reagents... pH-controlled coupling reactions
Implementation Method 2
organic solvent elution for cleaner mass spectra
Implementation Method 3
analysis by mass spectrometry... cleaner mass spectra
Data Source
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AI summary
The present invention provides a method for labelling one or more analytes in a sample, the method comprising: a) contacting the sample with one or more bifunctional linker reagents having the general formula Re1-L1-Re2, wherein Re1 is a first reactive group, L1 is a linker moiety and Re2 is a second reactive group, wherein Re1 reacts with an analyte to form a modified analyte; and b) contacting the sample with one or more mass labels, wherein Re2 of the bifunctional linker attached to the analyte reacts with a mass label to form a labelled analyte, wherein each mass label is relatable to an analyte by mass spectrometry.