Characterization of sphingosine-1-phosphate lyase activity by electrospray ionization-liquid chromatography/tandem mass spectrometry quantitation of (2E)-hexadecenal

Abstract

Sphingosine-1-phosphate (S1P) is a sphingolipid signaling molecule crucial for cell survival and proliferation. S1P-mediated signaling is largely controlled through its biosynthesis and degradation, and S1P lyase (S1PL) is the only known enzyme that irreversibly degrades sphingoid base-1-phosphates to phosphoethanolamine and the corresponding fatty aldehydes. S1PL-mediated degradation of S1P results in the formation of (2E)-hexadecenal, whereas hexadecanal is the product of dihydrosphingosine-1-phosphate (DHS1P) degradation. Fatty aldehydes can undergo biotransformation to fatty acids and/or alcohols, making them elusive and rendering the task of fatty aldehyde quantitation challenging. We have developed a simple, highly sensitive, and high-throughput protocol for (2E)-hexadecenal quantitation as a semicarbazone derivative by liquid chromatography-electrospray ionization-tandem mass spectrometry. The approach was applied to determining S1PL activity in vitro with the ability to use as low as 0.25μg of microsomal protein per assay. The method is also applicable to the use of total tissue homogenate as the source of S1PL. A correction for (2E)-hexadecenal disappearance due to its biotransformation during enzymatic reaction is required, especially at higher protein concentrations. The method was applied to confirm FTY720 as the inhibitor of S1PL with an IC₅₀ value of 52.4μM.

Figure 1

Chemical synthesis of (2E)-hexadecenal semicarbazone.

Figure 2

Positive product ion mass spectra of fatty aldehyde semicarbazone derivatives. (A) (2E)-Hexadecenal semicarbazone product ions. (B) cis-11-Hexadecenal semicarbazone product ions. (C) Hexadecanal semicarbazone product ions. (D) Proposed pathways for product ion formation during (2E)-hexadecenal semicarbazone collision-induced decomposition. Refer to text for the parameters of declustering, collision-induced decomposition, and collision cell exit potentials.

Figure 3

Positive ion ESI-LC/MS/MS analysis of semicarbazone derivatives of cis-11-hexadecenal, (2E)-hexadecenal, (2E)-d₅-hexadecenal, and hexadecanal. The MRM based on the [M-17]⁺ transition is shown for the simplicity. The MRM profile for (2E)-d₅-hexadecenal semicarbazone is shown as a dotted line.

Figure 4

Time- and pH-dependent formation of (2E)-hexadecenal semicarbazone. (2E)-hexadecenal (20 µM) was derivatized with 5 mM semicarbazide in methanol containing different concentration of formic acid for up to 6 h. Aliquots were withdrawn at the indicated time points and immediately analyzed by ESI-LC/MS/MS (10 pmol on-column for each data point). Data represent the average of the peak area corresponding to [M-17]⁺ transition ± S.E. of three independent measurements.

Figure 5

Microsomal association of S1PL activity. Mouse liver microsomes were isolated from a total liver homogenate by ultracentrifugation. The S1PL reaction was performed for 20 min with 40 µM S1P and 5 µg total homogenate, cytosolic, or microsomal protein. Lipid extraction and derivatization was performed as described in the text. Data are presented as the mean ± S.E. of three independent experiments.

Figure 6

Disappearance of (2E)-hexadecenal during enzymatic reaction as a function of time and protein concentration. (2E)-Hexadecenal (20 pmol) was incubated with the indicated amount of mouse liver microsomal protein in the reaction buffer. At the indicated time points, proteins were denatured by addition of methanol and chloroform (2:1, v/v). (2E)-d₅-Hexadecenal (20 pmol) was added as the internal standard before lipid extraction and phase separation. Total lipid extracts were derivatized with 5 mM semicarbazide in 5% formic acid in methanol. Data represent the average ± S.E. of three different experiments.

Figure 7

The linearity of S1PL reaction. The reaction was performed for 20 min with 40 µM S1P as substrate and variable amounts of liver microsomal protein. The reaction was stopped by lipid extraction with the addition of 20 pmol (2E)-d₅-hexadecenal as the internal standard. Total lipid extracts were derivatized with 5 mM semicarbazide in 5% formic acid in methanol for 2 h at 40°C and analyzed by ESI-LC/MS/MS. Data are presented as the mean ± S.E. of three independent experiments and were corrected for the disappearance of (2E)-hexadecenal during the enzymatic reaction.

Figure 8

S1PL activity as a function of substrate concentration. The S1PL reaction was performed for 20 min with variable amounts of S1P and 5 µg liver microsomal protein. The reaction was stopped by lipid extraction with the addition of 20 pmol (2E)-d₅-hexadecenal as the internal standard. Total lipid extracts were derivatized with 5 mM semicarbazide in 5% formic acid in methanol for 2 h at 40°C and analyzed by ESI-LC/MS/MS. Data are presented as the mean ± S.E. of three independent experiments.

Figure 9

FTY720 but not FTY720P inhibits S1PL activity in vitro. The S1PL reaction was performed for 20 min with 40 µM S1P as substrate and 5 µg microsomal protein, in the presence or absence of 0.57 mM pyridoxal-5’-phosphate (P5P). FTY720 (30 µM) or FTY720P (30 µM) was added together with S1P. The reaction was stopped by lipid extraction with the addition of 20 pmol (2E)-d₅-hexadecenal as the internal standard. Total lipid extracts were derivatized with 5 mM semicarbazide in 5% formic acid in methanol for 2 h at 40°C and analyzed by ESI-LC/ MS/MS. Data are presented as the mean ± S.E. of three independent experiments. *** -p<0.001; N.S. – not significant.

Figure 10

Dose-dependent inhibition of S1PL activity by FTY720. The S1PL reaction was performed for 20 min with 40 µM S1P as a substrate and 25 µg total mouse liver homogenate protein, in the presence of 0.57 mM pyridoxal-5’-phosphate (P5P). FTY720 was added together with S1P. The reaction was stopped by lipid extraction with the addition of 20 pmol (2E)-d₅-hexadecenal as the internal standard. Total lipid extracts were derivatized with 5 mM semicarbazide in 5% formic acid in methanol for 2 h at 40°C and analyzed by ESI-LC/MS/MS. Data are presented as the mean ± S.E.M. of three independent experiments. IC₅₀ was calculated using GraphPad Prism 5.0 data analysis tool.