E. coli total lipid extract was purchased from Avanti Polar Lipids (Alabaster, AL, USA) and analyzed on the LTQ Orbitrap XL instrument in negative ion mode. A solution of the total lipid concentration of 2.5 μg/ml in 7.5 mM ammonium acetate in choloroform/methanol/2-propanol (1/2/4, v/v/v) was infused into the mass spectrometer by TriVersa robotic ion source using a chip with the diameter of spraying nozzles of 4.1 μm. To produce the spectra dataset, the extract was analyzed in several independent experiments: experiment I, eight acquisitions under the unit mass resolution (R) settings using ion trap (IT) to acquire both MS and MS/MS spectra; experiment II, six acquisitions with R = 7,500 for MS spectra (Orbitrap) and unit resolution for MS/MS spectra (IT); experiment III, four acquisitions with R = 30,000 for MS spectra (Orbitrap) and unit resolution for MS/MS spectra (IT); experiment IV, four acquisitions with R = 100,000 for MS spectra (Orbitrap) and unit resolution for MS/MS spectra (IT); experiment V, seven acquisitions with R = 100,000 for MS spectra (Orbitrap) and R = 15,000 for MS/MS spectra (Orbitrap).
In the experiments I to IV, each acquisition produced approximately 33 MS and 330 MS/MS spectra; in the experiment V, 10 MS and 100 MS/MS spectra were acquired. To reduce undersampling, in the experiment V, acquisition of MS/MS spectra was navigated by the inclusion list compiled from 40 masses of plausible PE, PG and PA precursors A list of molecular lipid species was produced by manual interpretation of spectra acquired in the experiment V with requested mass tolerance of better than 3 ppm for precursors and 5 ppm for specific fragment ions. Only lipid species identified in at least four out of seven replicated analyses were included.
Spectra acquired in each of the experiments I to IV were further processed by LipidXplorer to produce corresponding MasterScan files. We used the dataset from the experiment I for comparative benchmarking of LipidXplorer against LipidQA and LipidSearch programs. Since LipidQA and LipidSearch do not align the spectra from replicated analyses, each acquisition was processed independently and then a non-redundant list of all identified lipid species was compiled.
In the experiments I to IV, each acquisition produced approximately 33 MS and 330 MS/MS spectra; in the experiment V, 10 MS and 100 MS/MS spectra were acquired. To reduce undersampling, in the experiment V, acquisition of MS/MS spectra was navigated by the inclusion list compiled from 40 masses of plausible PE, PG and PA precursors A list of molecular lipid species was produced by manual interpretation of spectra acquired in the experiment V with requested mass tolerance of better than 3 ppm for precursors and 5 ppm for specific fragment ions. Only lipid species identified in at least four out of seven replicated analyses were included.
Spectra acquired in each of the experiments I to IV were further processed by LipidXplorer to produce corresponding MasterScan files. We used the dataset from the experiment I for comparative benchmarking of LipidXplorer against LipidQA and LipidSearch programs. Since LipidQA and LipidSearch do not align the spectra from replicated analyses, each acquisition was processed independently and then a non-redundant list of all identified lipid species was compiled.
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