Abstract
A popular fragmentation technique for non-targeted analysis is called data-independent acquisition (DIA), because it provides fragmentation data for all analytes in a specific mass range. In this work, we demonstrated the strengths and weaknesses of DIA. Two types of chromatography (fractionation/3 min and hydrophilic interaction liquid chromatography (HILIC)/18 min) and three DIA protocols (variable sequential window acquisition of all theoretical mass spectra (SWATH), fixed SWATH and MSALL) were used to evaluate the performance of DIA. Our results show that fast chromatography and MSALL often results in product ion overlap and complex MS/MS spectra, which reduces the quantitative and qualitative power of these DIA protocols. The combination of SWATH and HILIC allowed for the correct identification of 20 metabolites using the NIST library. After SWATH window customization (i.e., variable SWATH), we were able to quantify ten structural isomers with a mean accuracy of 103% (91–113%). The robustness of the variable SWATH and HILIC method was demonstrated by the accurate quantification of these structural isomers in 10 highly diverse blood samples. Since the combination of variable SWATH and HILIC results in good quantitative and qualitative fragmentation data, it is promising for both targeted and untargeted platforms. This should decrease the number of platforms needed in metabolomics and increase the value of a single analysis.
Highlights
Fluctuations in the levels of metabolites have frequently been correlated to health, disease or response to treatment [1,2,3]
A more selective and comprehensive targeted approach can be realized by multiple reaction monitoring (MRM) high-resolution (MRMHR ) ( known as parallel reaction monitoring (PRM))
Our work demonstrates that the performance of data-independent acquisition (DIA) methodologies is highly dependent on the type of chromatography and the organization of the Q1 filters
Summary
Fluctuations in the levels of metabolites have frequently been correlated to health, disease or response to treatment [1,2,3]. A more selective and comprehensive targeted approach can be realized by MRM high-resolution (MRMHR ) ( known as parallel reaction monitoring (PRM)) In this mass spectrometry mode, a preselected precursor is isolated and fragmented followed by a high-resolution scan of all the produced product ions. The fragmentation data of DIA methodologies can be used for two purposes: identification and quantification This offers great potential for combined quantitative and qualitative platforms, which are increasingly implemented in the field of metabolomics [17]. The high-throughput platform consisted of a fractionation-based separation using three short chromatographic columns and an analysis time of 3 min [27] These platforms differ in separation efficiency and peak width and thereby reflect the demands of different types of chromatography in terms of DIA analyses. DIA methodologies using different separation mechanisms should demonstrate the usefulness and the limitations of DIA methodologies in terms of quantification and identification
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