Targeted MS3 Quantitation for Multiplexed Mass Spectrometry
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Solution Overview
Problem
Current methods for multiplexed targeted mass spectrometry face challenges in accurately quantifying specific target analytes in complex mixtures due to 'ratio distortion' from co-eluting contaminants and the reliance on expensive internal standards or data-dependent acquisitions, which can lead to incomplete analyte identification and quantification.
Innovation Solution
The method involves performing targeted MS3 analyses during retention time-scheduled acquisition segments, accompanied by periodic MS2 analyses, to determine the relative quantity of isobaric tag-labeled target analytes by spectral matching and quality scoring, reducing reliance on internal standards and data-dependent acquisitions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If isobaric tag-labeled analytes are multiplexed and analyzed via LC-MS, then sample throughput is increased, but ratio distortion occurs due to co-eluting contaminants leading to inaccurate quantitation
Solution Approach 1:
The mass spectrometry analysis is segmented into multiple stages: MS1 survey scans identify precursor ions, MS2 analyses provide preliminary filtering, and targeted MS3 analyses perform final specific detection. This segmentation allows the system to maintain high throughput by efficiently processing multiple samples while achieving accurate quantitation through staged filtering of contaminant ions at each mass analysis level.
2Reliability
If targeted MS3 analysis is performed using data dependent acquisition, then contaminant analytes are filtered, but data dependent acquisition leads to incomplete analyte identification and quantification
Solution Approach 1:
The system performs preliminary MS1 survey scans and MS2 analyses before the targeted MS3 analysis. These preliminary actions identify precursor ions and their fragmentation patterns, allowing the subsequent MS3 analysis to be targeted specifically at relevant analytes while maintaining comprehensive coverage. This preliminary action ensures that no potential analytes are missed while still achieving effective contaminant filtering.
3Measurement precision
If internal standards are used for quantitation, then accuracy is improved, but experimental complexity and cost increase
Solution Approach 1:
The mass spectrometry system performs self-calibration and self-quantitation by using the known m/z values and fragmentation patterns of isobaric tag-labeled analytes identified in the MS1 survey scans. The system automatically determines relative quantities based on the intensity of reporter ions in the targeted MS3 analysis, eliminating the need for external internal standards while maintaining quantitation accuracy through its inherent mass analysis capabilities.
4Measurement precision
If targeted MS3 analysis is performed during retention time-scheduled acquisition segments, then quantitation accuracy with low ratio distortion is achieved, but acquisition time increases
Solution Approach 1:
The system performs targeted MS3 analyses periodically during retention time-scheduled acquisition segments, interspersed with MS2 analyses. This periodic action allows the system to dedicate specific time windows to high-precision MS3 measurements when target analytes are expected to elute, while maintaining overall throughput by using the periodic MS2 analyses for broader screening and by efficiently scheduling the MS3 measurements within the chromatographic runtime.
Data Source
AI summary
A method of performing targeted multiplexed mass spectrometry includes performing, at a mass spectrometer, a targeted MS3 analysis of an isobaric tag-labeled target analyte included in a multiplex sample eluting from a column. The targeted MS3 analysis is performed during an acquisition segment scheduled based on an expected retention time of the isobaric tag-labeled target analyte. The method further includes performing, during the acquisition segment, a plurality of MS2 analyses of product ions derived from components included in the multiplex sample and eluting from the column. The method further includes determining, based on MS3 mass spectra acquired by the targeted MS3 analysis and MS2 mass spectra acquired by the plurality of MS2 analyses, a relative quantity of the isobaric tag-labeled target analyte in the multiplex sample.