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

Abstract

Sphingolipids are a class of lipids that share a sphingoid base backbone. They exert various effects in eukaryotes, ranging from structural roles in plasma membranes to cellular signaling. De novo sphingolipid synthesis takes place in the endoplasmic reticulum (ER), where the condensation of the activated C₁₆ fatty acid palmitoyl-CoA and the amino acid L-serine is catalyzed by serine palmitoyltransferase (SPT). The product, 3-ketosphinganine, is then converted into more complex sphingolipids by additional ER-bound enzymes, resulting in the formation of ceramides. Since sphingolipid homeostasis is crucial to numerous cellular functions, improved assessment of sphingolipid metabolism will be key to better understanding several human diseases. To date, no assay exists capable of monitoring de novo synthesis sphingolipid in its entirety. Here, we have established a cell-free assay utilizing rat liver microsomes containing all the enzymes necessary for bottom-up synthesis of ceramides. Following lipid extraction, we were able to track the different intermediates of the sphingolipid metabolism pathway, namely 3-ketosphinganine, sphinganine, dihydroceramide, and ceramide. This was achieved by chromatographic separation of sphingolipid metabolites followed by detection of their accurate mass and characteristic fragmentations through high-resolution mass spectrometry and tandem-mass spectrometry. We were able to distinguish, unequivocally, between de novo synthesized sphingolipids and intrinsic species, inevitably present in the microsome preparations, through the addition of stable isotope-labeled palmitate-d₃ and L-serine-d₃. To the best of our knowledge, this is the first demonstration of a method monitoring the entirety of ER-associated sphingolipid biosynthesis. Proof-of-concept data was provided by modulating the levels of supplied cofactors (e.g., NADPH) or the addition of specific enzyme inhibitors (e.g., fumonisin B₁). The presented microsomal assay may serve as a useful tool for monitoring alterations in sphingolipid de novo synthesis in cells or tissues. Additionally, our methodology may be used for metabolism studies of atypical substrates - naturally occurring or chemically tailored - as well as novel inhibitors of enzymes involved in sphingolipid de novo synthesis.

Keywords: ceramides; mass spectrometry; serine palmitoyltransferase; sphingolipid de novo synthesis; stable-isotope labeling.

FIGURE 1

Schematic overview of the sphingolipid de novo synthesis located in the ER. Serine palmitoyltransferase (SPT) catalyzes the condensation of palmitoyl-CoA and L-serine. The product of this reaction, 3-ketosphinganine (3KS), is further NADPH-dependently reduced to sphinganine (dihydrosphingosine, d18:0 Sph) by 3-ketosphinganine reductase (KDSR). Ceramide synthases (CerS) couple fatty acyl-CoAs to the amino group of the long-chain base d18:0 Sph. This leads to the formation of dihydroceramides (dhCer), differing in the chain length of the amide-bound fatty acid. The final step is the introduction of a double bond between carbons C-4 and C-5 mediated by dihydroceramide desaturase (DEGS) under NAD[P]H consumption. The formed ceramides (Cer) are subsequently shuttled to the Golgi apparatus for further metabolism. The cell image (left) was taken from https://smart.servier.com (freely accessible).

FIGURE 2

SPT-catalyzed formation of 3KS-d₅ from the in vitro assay substrates palmitate-d₃ and L-serine-d₃. (Top) Chemical structure, formula, and exact mass of the protonated molecular ion [M+H]⁺ of 3KS-d₅ generated during electrospray ionization. Dashed black line represents the mass transition used for selected reaction monitoring (SRM). (A) Overlay of extracted ion chromatograms (EIC) of the microsomal de novo synthesis assay using palmitate-d₃ and serine-d₃ as substrates (blue) compared to the negative control omitting palmitate-d₃ (green). (B) Mass spectrum extracted from the EIC in the retention time window of eluting 3KS-d₅ with annotation of the molecular ion peak and the calculated mass error. (A,B) Analysis was conducted on a quadrupole time-of-flight mass spectrometer (QTOF MS). (C) Overlay of SRM analyses of assay (blue) compared to negative control sample (green) performed with a triple quadrupole mass spectrometer (TQ MS). t_R, retention time. (A) In the t_R range of 8–10 min, relatively abundant unidentified matrix components (m/z: 305.2211 ≤ × ≤ 305.4211) eluted from both the assay and negative control samples. (C) Concordant matrix signals were diminished, with increased specificity achieved by analyzing compound-specific fragmentation.

FIGURE 3

Proposed catalytic cycle for serine palmitoyltransferase (SPT) with d₃-palmitoyl-CoA and L-serine-d₃ being the substrates. This scheme was modified from A. H. Merrill, Jr. (Merrill, 2011). An internal aldimine (Schiff base) formed between the cofactor pyridoxal 5’-phosphate (PLP) and an active site lysine residue (Enz-Lys-PLP, upper left) is replaced by the external aldimine through the incoming L-serine-d₃. Binding of d₃-palmitoyl-CoA results in transfer of serine α-deuterium to an enzymatic lysine residue (dashed box). The reaction proceeds as shown with free CoA, CO₂, and 3-ketosphinganine-d₅ (3KS-d₅) are released as products.

FIGURE 4

De novo formation of d18:0 Sph-d₅ from 3KS-d₅ in the in vitro assay. (Top) Chemical structure, formula, and exact mass of the protonated molecular ion [M+H]⁺ of d18:0 Sph-d₅ generated during electrospray ionization. Dashed black line represents the mass transition used for selected reaction monitoring (SRM). (A) Overlay of extracted ion chromatograms (EIC) of the microsomal de novo synthesis assay using palmitate-d₃ and serine-d₃ as substrates (blue) compared to the negative control omitting palmitate-d₃ (green). (B) Mass spectrum extracted from the EIC in the retention time window of eluting d18:0 Sph-d₅ with annotation of the molecular ion peak and the calculated mass error. (A,B) Analysis was conducted on a quadrupole time-of-flight mass spectrometer (QTOF MS). (C) Overlay of SRM analyses of assay (blue) compared to negative control sample (green) performed with a triple quadrupole mass spectrometer (TQ MS). t_R, retention time. (A) In the t_R range of 8.5–10 min, relatively abundant unidentified matrix components (m/z: 307.2367 ≤ × ≤ 307.4367) eluted from both the assay and negative control samples. (C) Concordant matrix signals were diminished, with increased specificity achieved by analyzing compound-specific fragmentation.

FIGURE 5

Formation of Cer d18:0-d₅/16:0-d₃ from de novo formed d18:0 Sph-d₅ and assay substrate palmitate-d₃. (Top) Chemical structure, formula, and exact mass of the protonated molecular ion [M+H]⁺ of Cer d18:0-d₅/16:0-d₃ generated during electrospray ionization. Dashed black lines represent the mass transitions used for multiple reaction monitoring (MRM). (A) Overlay of extracted ion chromatograms (EIC) from the microsomal de novo synthesis assay using palmitate-d₃ and serine-d₃ as substrates (blue), compared to the negative control omitting palmitate-d₃ (green). (B) Mass spectrum extracted from the EIC in the retention time window of eluting Cer d18:0-d₅/16:0-d₃ with annotation of the molecular ion peak and the calculated mass error. (A,B) Analysis was conducted on a quadrupole time-of-flight mass spectrometer (QTOF MS). (C) MRM analyses of assay (blue) compared to negative control sample (green) performed with a triple quadrupole mass spectrometer (TQ MS). For the negative control, only the mass transition with highest intensity (m/z 548.6 → 530.6) is shown. The arrow indicates the retention time (t_R) of Cer d18:0-d₅/16:0-d₃. (A) At t_R = 15 min, a relatively abundant unidentified compound (m/z: 548.4852 ≤ x ≤ 548.6852) eluted that was present in both assay and negative control samples. (C) Analysis of two analyte-specific fragmentations allowed for unambiguous discrimination between Cer d18:0-d₅/16:0-d₃ and matrix components.

FIGURE 6

Detection of Cer d18:1-d₅/16:0-d₃ as the terminal product of the sphingolipid de novo synthesis assay. (Right) Chemical structures, formulas and exact masses of the dehydrated, protonated molecular ions [M-H₂O+H]⁺ of Cer d18:1-d₅/16:0-d₃ and the unlabeled analog Cer d18:1/16:0, generated during electrospray ionization. Dashed black lines represent the mass transitions used for selected reaction monitoring (SRM). (Left) SRM analyses of assay sample (addition of palmitate-d₃ and serine-d₃, blue), negative control sample (omission of palmitate-d₃, green) and external Cer d18:1/16:0 standard (red) were performed with a triple quadrupole mass spectrometer (TQ MS). The arrow indicates the retention time of Cer d18:1/16:0 (labeled and unlabeled).

FIGURE 7

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-d₅ (first column), d18:0 Sph-d₅ (second column), Cer d18:0-d₅/16:0-d₃ (C16 dhCer-d₈, third column) and Cer d18:1-d₅/16:0-d₃ (C16 Cer-d₈, 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 B₁ (FB₁, 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.

FIGURE 8

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 B₁ (FB₁, 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-d₅ and d18:0 Sph-d₅ 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-d₅/16:0-d₃ (C16 dhCer-d₈) and Cer d18:1-d₅/16:0-d₃ (C16 Cer-d₈), 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.