Optimization of mass spectrometric parameters for a data dependent acquisition (DDA) experiment is essential to increase the MS/MS coverage and hence increase metabolite identifications in untargeted metabolomics. We explored the influence of mass spectrometric parameters including mass resolution, radio frequency (RF) level, signal intensity threshold, number of MS/MS events, cycle time, collision energy, maximum ion injection time (MIT), dynamic exclusion, and automatic gain control (AGC) target value on metabolite annotations on an Exploris 480-Orbitrap mass spectrometer. Optimal annotation results were obtained by performing ten data dependent MS/MS scans with a mass isolation window of 2.0 m/z and a minimum signal intensity threshold of 1 × 104 at a mass resolution of 180,000 for MS and 30,000 for MS/MS, while maintaining the RF level at 70%. Furthermore, combining an AGC target value of 5 × 106 and MIT of 100 ms for MS and an AGC target value of 1 × 105 and an MIT of 50 ms for MS/MS scans provided an improved number of annotated metabolites. A 10 s exclusion duration and a two stepped collision energy were optimal for higher spectral quality. These findings confirm that MS parameters do influence metabolomics results, and propose strategies for increasing metabolite coverage in untargeted metabolomics. A limitation of this work is that our parameters were only optimized for one RPLC method on single matrix and may be different for other protocols. Additionally, no metabolites were identified at level 1 confidence. The results presented here are based on metabolite annotations and need to be validated with authentic standards.
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