Comparative analysis of SP3 and S-Trap sample preparation protocols for proteomic profiling associated with somatic embryogenesis efficiency in Olea europaea L.

Somatic embryogenesis (SE) is an asexual plant propagation technique in which embryo-like structures are generated from somatic cells. It is a powerful tool for clonal propagation, genetic transformation, and germplasm conservation, but not all species respond efficiently to SE, limiting its application. To overcome this recalcitrance, recent studies have investigated the physiological, molecular, and cytological processes associated with SE in different plant species. Proteomics has emerged as a high-throughput approach to investigate global protein dynamics, and it has been increasingly applied to characterise SE-related molecular mechanisms. However, even using these large-scale proteomic strategies, the impact of sample preparation on data quality has never been systematically demonstrated. Here, we compared, for the first time, two sample preparation strategies for the analysis of SE cultures by liquid chromatography-tandem orbitrap mass spectrometry (LC-MS/MS), including (i) single-pot solid-phase-enhanced sample preparation (SP3), performed on an OT-2 automated platform, and (ii) the Suspension Trapping (S-Trap). For this purpose, we analysed the proteome of Olea europaea embryogenic lines with high and low efficiencies, maintained through secondary SE. More than 9,000 protein groups were identified in each method, most of which (7,735 proteins) were shared between workflows, while only a small proportion were uniquely detected by either workflow. Protein quantification was also highly consistent, with 8,454 and 8,365 protein groups quantified for SP3 and S-Trap, respectively, and 7,075 in common. Both workflows achieved comprehensive proteome coverage, with SP3 capturing additional functional categories, such as mitochondrial components, membrane-associated proteins, and vesicle trafficking. S-Trap provided complementary but comparatively more restricted coverage, while both approaches consistently captured key biological processes related to metabolism and intracellular organisation. Overall, the results demonstrate that SP3 and S-Trap workflows provide highly consistent and complementary proteomic coverage, supporting their combined or alternative use for comprehensive proteomic studies of SE.