LX Reference Raa 2009
Materials and Methods
Lipids were recovered by a two-step methanol/chloroform extraction as described (53). Settings that ensure accurate and robust quantification have been established in our previous work regarding sample amount, extraction procedure and mass spectrometric measurements (34,53,54). Basically, 900 000 cells (harvested following 4, 24 and 48 h of incubation) were taken up in 180 µL of 155 mm ammonia carbonate and 100 pmol of each lipid standard was added (PE-O 20:0/-O 20:0; PC-O 18:0/-O18:0; SM 35:1; Cer 35:1; GlcCer 30:1) and then extracted with 900 µL chloroform/methanol (10:1), while the water phase was further extracted with chloroform/methanol (2:1). Ceramides, hexosylceramides and lactosylceramides were partitioned into the phase with 10:1 choloroform/methanol content, while other lipids, including Gb3, were enriched in the 2:1 phase. The extracts were dissolved in chloroform/methanol/2-propanol 1/2/4 (v/v/v) containing 7.5 mm ammonium acetate. Before the MS analysis, samples were vortexed thoroughly and centrifuged for 2 min at 14 000 rpm on a Minispin centrifuge (Eppendorf, Hamburg, Germany). Samples were then loaded onto 96 well plates and sealed with aluminum foil. The lipids were analyzed by MS as described (34) using a LTQ Orbitrap (Thermo Fisher Scientific, Bremen, Germany) instrument equipped with a robotic nanoflow ion source NanoMate HD (Advion BioSciences Ltd, Ithaca, NJ) with 4.1 µm nozzle diameter chip NanoMate HD controlled by Chipsoft 6.4. software (Advion Biosciences) and operated at the ionization voltage of 0.95 kV and gas pressure 1.25 psi in positive ion mode or -1.05 kV in negative ion mode. MS survey scans were acquired using the Orbitrap analyzer operated under the target mass resolution of 100 000 [full width at half maximum (FWHM), defined at m/z 400]. MS/MS experiments on a LTQ Orbitrap instrument were performed using collision-induced dissociation in the linear ion trap or, where specified, in the intermediate C-trap (termed CID and HCD, respectively). Precursors were selected within the m/z window of 1.5 amu (precursor m/z± 0.75 amu). Acquired MS and MS/MS spectra were interpreted using proprietary software, which identified and quantified the lipid species (Herzog et al., 56th ASMS Conference on Mass Spectrometry 2008). In the 10:1 extracts, the PC, PE and SM species were quantified using the standards for their own lipid classes (described earlier), whereas the relative amounts ('abundance’) of Gb3 species (no standard available) were obtained by using the sum of the intensities for the standards of PC, PE and SM. In the 2:1 extracts, Cer and HexCer species were quantified by using the standards for their own lipid classes, whereas the abundance of LacCer (no standard available) was obtained by using the HexCer standard. Thus, as no standards were available for Gb3 and LacCer, we had to show ‘abundance,’ obtained as described here, whereas other species are reported as pmol per 106 cells. The data are reported as mean ± SD based on analysis of three cell extracts analyzed in duplicates (n = 6). Subsequent principal component analysis and t-test analysis were performed by MarkerView software (MDS Sciex, Concord, Canada).
