Maldi plate

MALDI-TOF MS was performed using an UltrafleXtreme MALDI-TOF/TOF mass spectrometer (Bruker Daltonik; Bremen, Germany). The TOF was calibrated using a peptide mixture (Bruker Daltonik) before acquisition of the sample spectra. The dried lipid extracts were reconstituted in 200 μl of CHCl₃/MeOH (2:1, v/v). Equal volume (1–2 μl) of lipid extract and MALDI matrix solution (0.5 M 2,5-dihydroxybenzoic acid, DHB, in methanol containing 0.1% trifluoroacetic acid) was mixed in a 0.6-ml microcentrifuge tube. Then, 1 μl of the mixture was spotted onto a MALDI plate (Bruker Daltonik). The mass spectra were acquired over the m/z range of 0–1,000 in both positive and negative ion Reflectron TOF modes. The laser power was adjusted to a point just above the ionization threshold of the sample, and the laser rate was set at 10 Hz with 1,000 laser shots per acquisition. Ten acquisitions were averaged for each individual sample.
Tandem mass spectrometry (MS/MS) analysis was performed on molecular species of interest (some major abundant lipid classes) using LIFT-TOF/TOF mode. The product ions characteristic head-group of the lipid was used to identify the lipid class. Fragment ions were generated by LIFT (laser ionization fragmentation technology) approach as described previously15 (link).

In 14 centres, the protein extraction procedure was performed by sonication as previously described^{11 (link),23 (link)} and the remaining laboratory applied Myco-EX protocol recommended by the manufacturer. Upon arrival in the laboratory, the tubes with the samples were centrifuged 5 min at 14,000 rpm and the pellet was resuspended in 300 µl of high-performance liquid chromatography (HPLC) or sterile Milli-Q water, after which they were heat inactivated at 95 °C for 30 min. After the addition of 900 µl of ethanol, the tubes were centrifuged for 2 min at 14,000 rpm and the supernatant was discarded. The pellet was dried for a few minutes at room temperature. Then, a spatula tip with 0.5 mm diameter glass or zirconia/silica beads (BioSpec products, USA) and 50 µl of acetonitrile were added. The tubes were vortexed for 10 s. In 14 laboratories, an additional step of 15 min sonication was performed. After this, 50 µl of formic acid were added and the tubes were vortexed again for 10 s. Finally, the samples were centrifuged for 2 min at 14,000 rpm and 1 µl of the supernatant was deposited by triplicate onto the MALDI plate (Bruker Daltonics, Bremen, Germany). After drying, 1 µl of HCCA (α-cyano-4-hydroxycinnamic acid) matrix was added to the spots and dried at room temperature.