Detection of phosphorylated sphingosine analogs. (A) LC-HRMS verification of the identity of the clickable sphingosine analogs used and detection of a phosphorylation product of ω-N 3 -sphingosine catalyzed by SphK1 in vitro . Lipid extracts from incubation of 1-N 3 -sphingosine or ω-N 3 -sphingosine with SphK1 and ATP were chromatographically separated by HPLC and analyzed with a quadrupole-time-of-flight mass spectrometer (QTOF MS) operating in the positive <t>electrospray</t> ionization mode <t>(ESI+).</t> Chemical structures and mass spectra of ionized ω-N 3 -sphingosine (left), 1-N 3 -sphingosine (middle), and phosphorylated ω-N 3 -S1P (right) are shown. No phosphorylation product of 1-N 3 -sphingosine could be detected. The retention time (t R ) of each substance analyzed is given as inset (top left) in the respective mass spectrum. (B) Detection of ω-N 3 -sphingosine 1-phosphate in Chang cells by LC-MS/MS. Chang cells were incubated with either 1-N 3 -sphingosine or ω-N 3 -sphingosine (10 μM) for 17 h. Lipid extracts were analyzed by selected reaction monitoring (SRM) after positive electrospray ionization (ESI+). As expected, no phosphorylation product could be detected for 1-N 3 -sphingosine (not shown). On the other hand, both ω-N 3 -sphingosine (upper chromatogram) and ω-N 3 -S1P (lower chromatogram) were present in lipid extracts of Chang cells stimulated with ω-N 3 -sphingosine. Assuming similar MS/MS responses for precursor and phosphorylation product, a conversion of about 20% occurred under the given experimental conditions. Peaks are labeled with retention time. Structural formulas and MS/MS fragmentations monitored are given as insets. CID, collision-induced dissociation.
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