Isobaric Labeling Reagent Sets for Multiplexed Peptide Quantitation
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Solution Overview
Problem
Current isobaric mass tagging systems for multiplexed quantitation of peptides by mass spectrometry are limited in multiplexing capacity due to the number of atoms available for isotopic substitution and the complexity of distinguishing reporter ions of the same mass, which complicates analysis and reduces sensitivity and throughput.
Innovation Solution
The use of isobaric labeling reagents with reporter groups having the same elemental composition and nominal mass but different isotopic substitutions, such as 13C and 15N, allows for higher resolution of reporter ions in mass spectrometry, enabling more samples to be analyzed within a single chromatographic run and increasing overall sample throughput.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If traditional isobaric mass tagging systems are used, then multiplexing capacity is limited by the number of atoms available for isotopic substitution, but increasing multiplexing capacity requires more complex isotopic substitutions which increase difficulty and cost
Solution Approach 1:
The patent changes the mass parameter of reporter ions by using different elemental compositions (e.g., varying numbers of 13C and 15N atoms) while maintaining isobaric relationships. This allows creation of labeling reagent sets with higher multiplexing capacity (e.g., 10-plex, 16-plex, 32-plex) without requiring increasingly complex isotopic substitutions on the same atoms, thereby resolving the contradiction between multiplexing capacity and complexity
2Adaptability or versatility
If reporter ions of the same mass are used, then multiplexing is limited, but resolving and distinguishing these reporter ions complicates mass spectrometry analysis
Solution Approach 1:
The patent varies the elemental composition parameters of reporter ions (different combinations of 13C and 15N atoms) to create distinct exact masses while maintaining nominal isobaric relationships. This enables mass spectrometry instruments to resolve and distinguish between reporter ions from different samples based on their precise mass differences, thereby increasing multiplexing capacity without excessively complicating detection and measurement
3Productivity
If more samples are analyzed in a single chromatographic run, then throughput increases, but analysis time and instrument time per sample may be reduced
Solution Approach 1:
The patent applies preliminary labeling action by attaching isobaric mass tags to peptides from multiple samples before mixing and chromatographic analysis. This allows all samples to be processed through a single chromatographic run simultaneously, increasing throughput while the preliminary labeling ensures that samples remain distinguishable throughout the analysis process
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 significantly enhances multiplexing capability, allowing for a greater number of samples to be analyzed efficiently, reducing analysis time, and improving the ability to directly compare data across samples, while maintaining sensitivity and throughput.
Implementation Method 1
Isobaric mass tagging or labeling is a method for determining the relative abundance of a given peptide across multiple samples by mass spectrometry
Implementation Method 2
The labeled peptides are then subjected to liquid chromatography and electrospray ionization mass spectrometry (LC-ESI-MS/MS)
Data Source
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AI summary
Disclosed herein are isobaric labeling reagent sets useful for multiplexed quantitation of peptides. The isobaric labeling reagent sets include a collection of at least two isobaric labeling reagents having first and second reporter groups with the same nominal mass but different isotopic substitutions and consequently different exact masses. Mass spectrometric analysis of the labeled samples is performed using a mass analyzer, such as an Orbitrap mass analyzer, capable of adequately resolving the ions of the first and second reporter groups. Reagent sets of the foregoing description may provide a degree of multiplexing in reporter ion quantitation experiments that is expanded relative to conventional labeling reagent sets, thereby reducing the number of chromatographic runs required for analysis and improving sample throughput.