Comparison of Full-Scan, Data-Dependent, and Data-Independent Acquisition Modes in Liquid Chromatography–Mass Spectrometry Based Untargeted Metabolomics

Abstract

Full-scan, data-dependent acquisition (DDA), and data-independent acquisition (DIA) are the three common data acquisition modes in high resolution mass spectrometry-based untargeted metabolomics. It is an important yet underrated research topic on which acquisition mode is more suitable for a given untargeted metabolomics application. In this work, we compared the three data acquisition techniques using a standard mixture of 134 endogenous metabolites and a human urine sample. Both hydrophilic interaction and reversed-phase liquid chromatographic separation along with positive and negative ionization modes were tested. Both the standard mixture and urine sample generated consistent results. Full-scan mode is able to capture the largest number of metabolic features, followed by DIA and DDA (53.7% and 64.8% respective features fewer on average in urine than full-scan). Comparing the MS2 spectra in DIA and DDA, spectra quality is higher in DDA with average dot product score 83.1% higher than DIA in Urine­(H), and the number of MS2 spectra (spectra quantity) is larger in DIA (on average 97.8% more than DDA in urine). Moreover, a comparison of relative standard deviation distribution between modes shows consistency in the quantitative precision, with the exception of DDA showing a minor disadvantage (on average 19.8% and 26.8% fewer features in urine with RSD < 5% than full-scan and DIA). In terms of data preprocessing convenience, full-scan and DDA data can be processed by well-established software. In contrast, several bioinformatic issues remain to be addressed in processing DIA data and the development of more effective computational programs is highly demanded.

Effects of myocardial ischemia/reperfusion injury on plasma metabolomic profile during aging

“…Of note, we believe the reason of not detecting all the putative metabolites in MS/MS analysis may be related to less sensitivity of Data Dependent Acquisition (DDA) in detecting low abundance metabolites than full‐scan MS (Guo & Huan, 2020).…”

New software tools, databases, and resources in metabolomics: updates from 2020

“…A spatial metabolomics pipeline (metaFISH) that combined fluorescence in situ hybridization (FISH) microscopy and high-resolution atmospheric-pressure matrix-assisted laser desorption/ionization mass spectrometry to image host–microbe symbioses and their metabolic interactions (Geier et al 2020 ) was also reported. Another study that compared the full-scan, data-dependent acquisition (DDA), and data-independent acquisition (DIA) methods in HR LC–MS/MS based metabolomics to reveal that spectra quality is better in DDA with average dot product score 83.1% higher than DIA and the number of MS 2 spectra (spectra quantity) is larger in DIA (Guo & Huan, 2020a ). Furthermore, it was shown that DDA mode consistently generated fewer uniquely found significant features than full-scan and DIA modes (Guo & Huan, 2020b ).…”

Reproducible Untargeted Metabolomics Data Analysis Workflow for Exhaustive MS/MS Annotation

“…For DIA, all ions are sent into the collision cell for fragmentation, and deconvolution algorithms are used to connect the fragment ions to the parent compounds. However, DDA and DIA cover only a subset of the full scan features and the selected precursor ions may come from background instead of biologically relevant features 11 . In addition, DDA and DIA are designed for qualitative analysis instead of performing quantitative analysis with fragment ions 12 , because a compromise must be made between more scan time for high quality fragment ions and well-shaped chromatography for precursor ions.…”

“…Proposed solutions include time-staggered precursor ion lists as inclusion lists 13 or automated exclusion lists to cover more compounds during repeated DDA injections 14 . However, the sensitivity of DDA's precursor ions is comparable with full scan mass spectra 11 limiting the possibility to find extra precursor ions by DDA. As an alternative to DDA or DIA, targeted MS/MS is a straightforward method for qualitative and quantitative analysis of known compounds.…”