Making a shotgun lipidomics dataset. Experiments are repeated in several independent biological replicates for each studied phenotype. Each biological replicate is split into several samples from which lipids are extracted and extracts are independently analyzed by MS. Spectra acquired from the total lipid extract survey molecular ions of lipid precursors, which are subsequently fragmented in MS/MS experiments, yielding MS/MS spectra. Each spectrum is acquired in several scans that are subsequently averaged. A set of MS and MS/MS spectra is termed as an 'acquisition' and several acquisitions are performed continuously making a 'technical replicate'.