Sphingomyelin analysis

Total lipid extracts were subjected to mild base hydrolysis to hydrolyze phospholipids. The resulting mixture was then extracted with chloroform:methanol to obtain the intact sphingolipids [13]. The resulting lipid extract was dried under nitrogen and resuspended in 200 μl of 2-propanol/methanol/chloroform (4:2:1 v/v/v) containing 20 mM ammonium formate and 0.5 μM SM (d18:1/12:0) as internal standard. Samples were introduced into a TSQ Ultra triple quadrupole mass spectrometer (Thermo Scientific, Waltham, Massachusetts) using a nanomate chip–based nano-ESI source (Advion Biosciences, Ithaca, New York) operating in infusion mode and SM species were measured using precursor ion scanning of m/z 184 [14]. Abundances of SM molecular species were calculated using the lipid mass spectrum analysis (LIMSA) software (University of Helsinki) and are represented as a relative percent of the sum based upon their response values.