Mass Spectrometry Quantitation Using DDA-Guided DIA Deconvolution
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Solution Overview
Problem
Data-independent acquisition (DIA) in mass spectrometry faces challenges with multiplexed isobaric labeled samples, particularly due to the overlap of reporter ion signals from different precursors, which hinders the accurate quantitation of biomolecules like peptides and proteins, especially in single cell proteomics where a large number of samples need to be compared.
Innovation Solution
A method combining data-dependent acquisition (DDA) and DIA, where MS2 spectra are acquired by DDA to form a spectral library, and then used to deconvolute the relative abundances of biomolecules in DIA spectra by matching peaks and calculating total abundances of mass tags, allowing for accurate quantitation while minimizing missing values.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If data-independent acquisition (DIA) is used to analyze multiplexed isobaric labeled samples, then the number of missing values is reduced, but reporter ion signals from different precursors overlap making accurate quantitation difficult
Solution Approach 1:
The patent segments the complex DIA MS2 spectrum by identifying and separating fragment ion signals from different precursor ions. Each precursor's fragment ions are individually analyzed and assigned to specific biomolecules, effectively dividing the overlapping signal mixture into distinct quantifiable components.
Solution Approach 2:
The patent uses an intermediary computational approach that leverages prior knowledge from data-dependent acquisition (DDA) experiments and spectral libraries to deconvolute the overlapping reporter ion signals. This intermediary processing layer translates the complex overlapping DIA data into separable quantitation information.
2Measurement precision
If data-dependent acquisition (DDA) is used to select specific precursor ions for fragmentation, then the MS2 mass spectrum becomes simpler and more specific, but many precursor ions are not selected resulting in missing values
Solution Approach 1:
The patent merges the advantages of both DDA and DIA approaches. It uses DDA to generate spectral libraries for specific precursor identification, then combines this with DIA's comprehensive sampling of all precursors. The computational method integrates data from both acquisition modes to achieve both specificity and completeness.
Solution Approach 2:
The patent performs preliminary action by conducting DDA experiments beforehand to build spectral libraries containing expected fragment ion patterns. These pre-acquired reference data are then used during DIA analysis to identify and quantify biomolecules, ensuring comprehensive coverage without missing values.
3Productivity
If a large number of samples are analyzed using DIA, then throughput is increased, but the overlap of reporter ion signals from different precursors becomes more severe
Solution Approach 1:
The patent uses spectral libraries created from DDA experiments as templates or copies of expected fragment ion patterns. These reference spectra are then matched against the actual DIA data to identify and quantify biomolecules, enabling accurate analysis even when reporter ion signals overlap severely in high-throughput scenarios.
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 enables high-throughput analysis with low missing values, effectively addressing the overlap issue in DIA, thereby providing accurate quantitation of biomolecules across multiple samples, even in single cell proteomics applications.
Implementation Method 1
mass spectrometry for analysing samples of biomolecules
Implementation Method 2
The fragment ions are then mass analysed in an MS/MS (also termed MS2) scan
Implementation Method 3
as it elutes from a liquid chromatographic device
Data Source
AI summary
A mass spectrometry method comprises: providing a multiplexed sample comprising a mixture of biomolecule-containing samples respectively tagged with mass tags; acquiring MS2 spectra by data-dependent acquisition (DDA) of the multiplexed sample or another mass tagged mixture of the samples during chromatographic elution; acquiring MS2 spectra by data-independent acquisition (DIA) during the elution; forming a spectral library from the DDA MS2 spectra comprising a plurality of the MS2 spectra and the biomolecule retention times; matching fragment-ion peaks in the DIA MS2 spectra to fragment-ion peaks in the MS2 library spectra to find matched biomolecules; determining a total abundance for each matched biomolecule from the DIA MS2 spectra at each of a plurality of retention times; determining abundances of respective reporter ions from the DIA MS2 spectra at the plurality of retention times; and deconvoluting relative abundances of the biomolecules in each respectively tagged biomolecule-containing sample based on the determined abundances.