Journal: Frontiers in Cell and Developmental Biology

Article Title: Monitoring the Sphingolipid de novo Synthesis by Stable-Isotope Labeling and Liquid Chromatography-Mass Spectrometry

doi: 10.3389/fcell.2019.00210

Figure Lengend Snippet: Impact of cofactor supplementation and presence of enzyme inhibitors on de novo formation of deuterated sphingolipid metabolites in the microsomal assay. Selected reaction monitoring (SRM) using a triple quadrupole mass spectrometer (TQ MS) was conducted to investigate the formation of 3KS-d 5 (first column), d18:0 Sph-d 5 (second column), Cer d18:0-d 5 /16:0-d 3 (C16 dhCer-d 8 , third column) and Cer d18:1-d 5 /16:0-d 3 (C16 Cer-d 8 , fourth column) under different assay conditions. The in vitro assay reaction mixture was prepared as described in section Materials and Methods with the following modifications: omission of NADPH (first row), “standard conditions” (with NADPH, second row), supplemented with NADPH and NADH (third to fifth row), addition of ceramide synthase (CerS) inhibitor fumonisin B 1 (FB 1 , fourth row), and addition of serine palmitoyltransferase (SPT) inhibitor myriocin (Myr, fifth row). Signals that were not present in the corresponding negative controls were integrated and colored. d17:0 Sph (0.2 pmol injected on column) and Cer d18:1/17:0 (2 pmol injected on column), referred to as internal standards (IS) 1 and 2, respectively, were used to normalize areas under the curves (AUC). Corresponding relations are indicated as insets. Y axes are scaled differently, while x axes are maintained for each sphingolipid metabolite.

Article Snippet: Cer d18:0/16:0 (C16 dhCer), Cer d18:1/16:0 (C16 Cer), Cer d18:1/17:0 (C17 Cer), d18:0 Sph (dhSph) and d17:0 Sph were from Avanti Polar Lipids (Alabaster, United States).

Techniques: Mass Spectrometry, In Vitro, Injection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Monitoring the Sphingolipid de novo Synthesis by Stable-Isotope Labeling and Liquid Chromatography-Mass Spectrometry

doi: 10.3389/fcell.2019.00210

Figure Lengend Snippet: Intra-assay variability of the sphingolipid de novo synthesis assay. A total of 10 technical assay replicates were prepared, five of which were supplemented with NADPH and NADH (white bars and solid circles), while the remaining five replicates were additionally with fumonisin B 1 (FB 1 , gray bars and open circles). Assays were performed as described in the Materials and Methods section using identical batches of microsomes and reagents. Concentrations of de novo formed, deuterated sphingolipid metabolites in the final sample volume (100 μL) were quantified by HPLC-MS/MS using external calibration (concentration range: 0–1000 nM). To this end, 3KS-d 5 and d18:0 Sph-d 5 were quantified via d18:0 Sph, whereas calibration curves of Cer d18:0/16:0 and Cer d18:1/16:0 were used to quantify Cer d18:0-d 5 /16:0-d 3 (C16 dhCer-d 8 ) and Cer d18:1-d 5 /16:0-d 3 (C16 Cer-d 8 ), respectively. Cer d18:1/17:0 and d17:0 Sph were used as internal standards. Bars represent mean concentrations (detailed in the main text) ± SEM ( ∗∗∗ p < 0.001; n.s., non-significant; n = 5). Additionally, individual values of the replicates are depicted as circles.

Article Snippet: Cer d18:0/16:0 (C16 dhCer), Cer d18:1/16:0 (C16 Cer), Cer d18:1/17:0 (C17 Cer), d18:0 Sph (dhSph) and d17:0 Sph were from Avanti Polar Lipids (Alabaster, United States).

Techniques: Intra Assay, Tandem Mass Spectroscopy, Concentration Assay