Figure 2.

MALDI-ToF (matrix assisted laser desorption ionization-time of flight) mass spectra analysis of the PgSPT (Porphyromonas gingivalis serine palmitoyl transferase) reaction between L-serine and palmitoyl-CoA. Each assay, which contained 1μM enzyme, 100mM HEPES, pH 7.0, 250mM NaCl, 0.2mM 5,5′-dithiobis-2-nitrobenzoic acid, and 20mM L-serine or 250μM palmitoyl-CoA, was added dependent on samples. All reaction samples were eluted with 100% acetonitrile (ACN) by C4 zip-tip and mixed with α-Cyano-4-hydroxycinnamic acid matrix dissolved in 50% ACN within 0.25% trifluoroacetic acid. The spectrum was analyzed on positive ion mode in triplicates. (A) Observation of the product ketodihydrosphingosine (KDS) with m/z = 300.290 during a sweep of masses (m/z = 100 to 800 amu). (B–D) Negative controls. (E) Full assay with PgSPT, L-serine, and P-CoA with a mass range of m/z = 292 to 304. (F) Theoretical mass spectrum based on the KDS formula (M+H)+.