Abi 4000 qtrap

SAM, SAH, and Hcy metabolites were quantified based on the platform developed by Xu et al. [73 (link)] with the following modifications, which were specific for these metabolites. The LC-MS/MS system consisted of an ABI 4000 QTRAP tandem mass spectrometer employing an electrospray ion source (Turbo Ion Spray™) (SCIEX, Foster City, CA, USA) in positive ionization mode in combination with a LC-10AD LC system (Shimadzu, Kyoto, Japan) equipped with a SIL-HTC autosampler. The metabolites were separated on a SeQuant ZIC–pHILIC column (150 × 4.6 mm, 5 μm particle size) with guard column (SeQuant ZIC–pHILIC, 20 × 2.1 mm, 5 μm particle size). The column temperature was set at 45 °C and the flow rate was 0.4 mL/min. Good chromatographic separation was observed with a 13 min gradient program consisting of mobile phases solvent A (20 mM ammonium formate, pH3.5) and solvent B (100% acetonitrile). Elution commenced with 80% B for one min, a linear gradient from 80% B to 5% B for 5 min, followed by a linear gradient back to 80% lasting 2 min, then isocratic hold on 80% for another 5 min. MS parameters were set as follows: ion source temperature (450 °C), ion spray voltage (5000 V), curtain gas (25 psig), collision gas (8 psig), ion source gas 1 (20 psig), ion source gas 2 (20 psig) interface heater activated.

Sphingolipids were analyzed by LC/ESI-MS/MS based on the method described with some modifications as described below [62 (link)]. Lipids were extracted from cell pellets (equivalent to 600 μg to 1 mg of protein depending on the experiment) using an azeotrophic mix of isopropanol:water:ethyl acetate (3:1:6; v:v:v). Internal standards (50 pmol of d17 long-chain bases and C12 acylated sphingolipids) were added to samples at the onset of the extraction procedure. Extracts were separated on an Agilent 1100 HPLC system. Mobile phases were as described [62 (link)], but with 0.2% formic acid on a Phenomenex Luna C8 (3 μm) 2.1 mm ID × 5 cm column maintained at 60°C for separation of the sphingoid bases and 1-phosphates or a Supelco 2.1 mm ID x 5 cm NH2 column for acylated sphingolipids. The eluate was analyzed with an inline ABI 4000 Q Trap (SCIEX, Framingham, MA) mass spectrometer equipped with a turbo ion spray source. Mass spectrometry settings were set to compensate for differences in ionization efficiency of different acyl chain lengths. The peak areas for the different sphingolipid subspecies were compared with that of the internal standards with 13C isotopic signals compensated for where necessary. All data reported are based on monoisotopic mass and are represented as pmol/mg protein.