DBA Isobaric Tags for Multiplexed PTM Quantification
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Solution Overview
Problem
Current methods lack versatile and high-throughput tools for studying post-translational modifications (PTMs), particularly for identifying and quantifying the dynamic changes and crosstalk of PTMs, which are crucial for understanding physiological activities and human diseases.
Innovation Solution
Development of novel isobaric chemical tags, such as DiLeu-Biotin-Azide (DBA) tags, that enable simultaneous isobaric labeling, high-efficiency click chemistry conjugation, and selective purification or enrichment of PTMs, allowing for high-throughput quantitative MS/MS analysis of proteins with PTMs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional PTM analysis methods are used, then identification of PTMs can be achieved, but high-throughput quantitative analysis and multiplexed analysis of multiple PTMs are limited
Solution Approach 1:
The patent combines multiple functional modules into a single integrated DBA tag system: (1) isobaric labeling module for multiplexed quantification, (2) biotin enrichment module for selective purification, and (3) azide conjugation module for PTM-specific labeling. This merging enables simultaneous achievement of high-throughput analysis and multiplexed PTM detection without requiring multiple separate experimental workflows
Solution Approach 2:
The DBA tag platform serves multiple functions: it enables isobaric labeling for quantitative proteomics, affinity enrichment for PTM isolation, and chemoselective conjugation for specific PTM targeting. This multi-functionality allows a single platform to address diverse PTM analysis needs (oxidation, carbonylation, citrullination, etc.) without requiring separate specialized methods for each application
2Adaptability or versatility
If PTM dynamics and crosstalk are studied comprehensively, then understanding of physiological activities and diseases is improved, but lack of versatile tools limits the ability to perform such studies
Solution Approach 1:
The DBA tag platform provides universal applicability across multiple PTM types through the use of different alkyne-containing probes that can target various PTMs (oxidized cysteine, carbonylated proteins, citrullinated proteins). The same core DBA tag structure with isobaric labeling and biotin enrichment can be used for all these PTMs, enabling comprehensive study of PTM dynamics and crosstalk without requiring separate specialized tools for each PTM type
Solution Approach 2:
The system segments the PTM analysis process into distinct modular components: (1) probe selection and PTM-specific labeling, (2) isobaric tag incorporation for multiplexed quantification, and (3) biotin-based enrichment for selective purification. This segmentation allows flexible adaptation to different PTM study requirements while maintaining comprehensive analytical capability
3Measurement precision
If isobaric labeling is performed for quantitative analysis, then relative quantification can be achieved, but multiplexed analysis of many samples simultaneously is limited
Solution Approach 1:
The patent merges isobaric labeling capability with biotin enrichment capability in the DBA tag system. The isobaric tags incorporate biotin moieties, allowing simultaneous achievement of precise quantitative measurement and high-throughput sample multiplexing. This integration enables analysis of many more samples simultaneously compared to conventional isobaric labeling methods that lack the enrichment component
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
The DBA tags facilitate comprehensive analysis of PTMs, including cysteine oxidation, protein carbonylation, and citrullination, with high reporter ion yield and enrichment efficiency, enabling unambiguous localization of modification sites and relative quantification at reduced collisional energies.
Implementation Method 1
a conjugation moiety (including, but not limited to, an azide functional group to enable biorthogonal click chemistry)
Implementation Method 2
an enrichment moiety (including, but not limited to, a biotin moiety) for selective purification or enrichment such as through binding with streptavidin beads
Implementation Method 3
Mass spectrometry (MS)-based proteomics has gained great popularity in detecting and structurally defining covalent changes in a protein
Data Source
AI summary
The present invention provides improved cleavable biotin-containing isobaric tags for quantitative mass spectrometry proteomics, particularly useful in quantification of post-translational modifications (PTMs) and assessing cysteine containing proteins. The isobaric chemical tags, DiLeu-Biotin-Azide (DBA), consist of three “modules”: (1) a DiLeu reporter group for relative quantification using MS/MS; (2) a biotin moiety for selective enrichment via streptavidin beads; and (3) an azide functional group to enable biorthogonal click chemistry. The DBA tags could be used in a high-throughput quantitative pan-PTM analysis platform.