Isobaric Stable-Isotope Phosphorylated Reagent for Low-Noise MS Quantification
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Solution Overview
Problem
Existing stable isotope labeling strategies for protein quantification in proteomics face limitations in sensitivity, selectivity, chromatographic separation, and high reagent costs, restricting large-scale quantitative analysis.
Innovation Solution
Development of an isobaric stable isotope-containing phosphorylated protein labeling reagent with novel organophosphorus structures for selective labeling of peptides, allowing for improved MS detection through secondary MS analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional stable isotope labeling strategies are used for protein quantification, then protein identification and quantification can be achieved, but the sensitivity and measurement precision are limited due to noise in MS spectra
Solution Approach 1:
The patent segments the MS analysis into two stages: first MS for peptide identification and second MS for quantification. The labeling reagent is designed to produce reporter ions that are specifically detected in the second MS stage, separating the identification and quantification functions to reduce noise interference in the quantification process
Solution Approach 2:
The patent introduces an intermediary substance - the isobaric stable isotope-containing phosphorylated labeling reagent - which acts as a mediator between the peptide and the MS detection system. This reagent contains reporter ions that serve as intermediaries to transmit quantification information while being distinguishable from background noise through secondary MS analysis
2Reliability
If existing labeling reagents are used, then protein quantification is possible, but the selectivity and chromatographic separation performance are insufficient
Solution Approach 1:
The patent applies local quality by designing the labeling reagent with specific functional groups that selectively label certain peptide regions. The reagent structure includes a phosphorylated moiety that targets specific amino acid residues, providing localized and selective labeling that enhances reliability without requiring complex separation procedures
Solution Approach 2:
The patent changes the chemical parameters of the labeling reagent by incorporating stable isotopes and phosphorylated groups. These parameter changes enable the reagent to produce distinct mass spectral signatures that improve selectivity and reduce the need for complex chromatographic separation
3Productivity
If conventional labeling strategies are employed, then basic protein quantification can be performed, but the quantitative flux and large-scale analysis capability are restricted due to high reagent costs
Solution Approach 1:
The patent creates a universal labeling reagent that can be applied to multiple peptides and proteins simultaneously. The isobaric stable isotope-containing phosphorylated structure allows the reagent to function across different sample types and experimental conditions, enabling large-scale quantitative analysis with improved productivity while maintaining cost-effectiveness through versatile application
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
Enhances the accuracy and sensitivity of protein quantification by reducing noise in secondary MS spectra, enabling high coverage and repeatability in quantitative analysis of proteins in various samples.
Implementation Method 1
stable isotope chemical labeling technology based on biological mass spectrometry (bio-MS) plays an increasingly important role in the relative and absolute quantification of proteins
Implementation Method 2
The rapid development of bio-MS instruments plays an essential role in the application and research in protein identification, post-translational modification (PTM), and quantitative protein analysis
Implementation Method 3
Based on the organophosphorus labeling chemistry, the present disclosure synthesizes a series of isobaric stable isotope-labeled organophosphorus reagents with novel structures
Data Source
AI summary
An isobaric stable isotope-containing phosphorylated protein labeling reagent, and a preparation method and application thereof are provided. The protein labeling reagent is a phosphorylated dipeptide organophosphorus reagent labeled by a stable isotope such as deuterium-2, carbon-13, oxygen-18, or nitrogen-15. The preparation method includes: (1) preparation of an isobaric stable isotope-containing amino acid with N-terminal protection, (2) preparation of an isobaric stable isotope-containing amino acid activated ester Fmoc/Boc-R1-NHS with N-terminal protection, (3) preparation of an isobaric stable isotope-containing dipeptide, (4) preparation of an isobaric stable isotope-containing phosphite, (5) preparation of an isobaric stable isotope-containing phosphite dipeptide, and (6) preparation of a stable isotope-labeled N-phosphorylated amino acid activated ester. The present protein labeling reagent can realize the quantitative analysis of polypeptides, standard proteins, proteins in cells, and proteins in urine samples and blood samples, and has the advantages of good accuracy, high sensitivity, no interference of isotope effect and wide applicability.