Experimental approach followed for the analysis of membrane sphingolipidomes. Arabidopsis (A. thaliana; Col-0) leaves from 11-week-old plants were used to obtain the crude membrane fraction, microsomes (MIC). From this, VM and PM were purified. PM fractions were obtained by two methods, phase partitioning, and free-flow electrophoresis. DRM fractions were obtained from the PMs isolated by two-phase partitioning. Every membrane preparation was independently treated and extracted for sphingolipid analysis by HPLC/ESI-MS/MS. Thus, the 18 independent membrane preparations are named 18 biological replicates and are composed of six MIC preparations, three VM preparations, three PM preparations obtained by two-phase partitioning, three PM preparations obtained by FFZE, and three DRM preparations. Every biological replicate was used for one sphingolipid extraction and sphingolipid analysis (one technical replicate). However, only for one biological sample from each type of membrane, two extractions were performed (two technical replicates). Therefore, a total of 23 technical replicates were obtained as shown. The different statistical tests applied to the data are indicated.