Tsq 7000 triple quadruple mass spectrometer

Blood samples were collected from the two patients and from an unrelated healthy control and lipids were extracted using ethyl acetate/isopropanol extraction buffer (detailed in the online supplementary file). Sphingolipids were determined by LC-MS/MS performed on a TSQ 7000 triple quadruple mass spectrometer (Thermo Finnigan; Ringoes, New Jersey, USA) as described in.^{6 (link)} The amount of sphingolipids in each sample was normalised to phosphates.

Blood samples were taken from patient 3 with a homozygous truncating mutation in the SGPL1 gene at age 5 months and from 2 healthy adult controls (36-year old male, 32-year old female) within the same hour and were transported and processed simultaneously. Fibroblast cultures from patient 3 and from two healthy adult controls (44-year old male, 39-year old female) were derived from skin punch biopsies, taken at different times. They had similar passage times when they were grown to 90% confluence in Innsbruck and shipped and processed together at the biochemistry lab in New York. Two ml of RPMI were added to 100 ul of each blood sample. To extract lipids, 2 ml extraction buffer, ethyl acetate/isopropanol/water (60/30/10, v/v) were added. After the extraction, formic acid was added to the organic phase. The lipids were extracted from the blood by the extraction buffer once more. After 2 extractions, they were combined and dried down under N₂ gas stream. Sphingolipids were determined by LC-MS/MS performed on a TSQ 7000 triple quadruple mass spectrometer (Thermo Finnigan; Ringoes, NJ) as described (Bielawski et al., 2006 (link)). Skin fibroblasts were cultured to 90% confluence in DMEM medium containing 20% FBS before being harvested for sphingolipid profiling by LC-MS/MS.