Speedvac vacuum centrifuge

PGN was isolated essentially following a published procedure [29 ]. Briefly, biomass was harvested from 1 l of culture grown to the stationary phase by centrifugation (5000 g, 30 min, 4 °C), resuspended in 60 ml distilled water and transferred drop-wise into 65 ml of boiling 8% sodium dodecyl sulfate (SDS; Sigma) under constant stirring to lyse cells. The suspension was further boiled for 1 h, reduced to the former volume using a rotary evaporator, and stirred overnight. SDS was removed by several washing steps with distilled water, 60 ml, each, using an Optima L-100XP ultracentrifuge from Beckman Coulter (rotor Ti70, 35,000 rpm, 30 min, 40 °C) followed by dialysis against distilled water for 4 days at room temperature. For the total volume of 12 ml of that PGN solution, 200 μl of an α-amylase solution (24 mg ml^− 1; Sigma) were added and the mixture was incubated at 37 °C for 2 h under constant shaking. Further, 320 μl of pre-incubated Pronase E solution (10 mg ml^− 1 in 10 mM Tris-HCl, pH 7.5; Sigma) was added and incubated at 60 °C for 1.5 h. Preparations were washed, boiled for 1 h, washed again, and dried in a Speed Vac vacuum centrifuge (Thermo Fisher Scientific, Vienna, Austria).

Lipids were extracted from frozen frontal lobe specimens (50 ± 5 mg) by the Folch method [33 (link)] after steel bead-based homogenization in sterile deionized water using a TissueLyser (Qiagen, Valencia, CA). After evaporating the organic phase to dryness in a SpeedVac vacuum centrifuge (Thermo Fisher Scientific, Waltham, MA), the pellets were dissolved in 100 μL HPLC grade methanol and stored at −80°C.
Saturated α-Cyano-4-hydroxycinnamic acid (HCCA, Sigma Aldrich, St. Louis, MO) prepared in TA50 (Acetonitrile/0.1% trifluoroacetic acid (TFA) in water, 50:50 v/v) was used as matrix and mixed 1:1 (v/v) with lipid extract. 1 μl aliquots were spotted into a 384-well ground steel MALDI Target Plate (MTP 384) (Bruker Daltonics, Bremen, Germany) and air-dried. The samples were analyzed in the negative ion mode of an Ultraflextreme MALDI-time-of-flight (TOF)/TOF (Bruker Daltonics, Bremen, Germany). Spectra data with the mass range set to 60–3500 Da were collected with rasterizing across each well and acquiring 150000 shots/well. Flex Analysis and tandem Mass Spectrometry (MS/MS) were performed on parent ions and lipid ion identifications were made based on assignments in the LIPID MAPS database (http://www.lipidmaps.org/tools/index.html). Statistical analysis was performed using ClinProTools, Version 3.0.

Proteins in the eluate were analyzed on a 6 to 15% SDS-PAGE gradient gel. Each lane was diced, and gel pieces were put into 1.5-mL microcentrifuge tubes. The samples were prepared by adapting standard protocols for in-gel mass spectrometry sample preparation (84 (link)). Incubation with 10 mM dithiothreitol (DTT) was carried out for 45 min at 55°C in order to reduce disulfide bonds. Afterwards, carbamidomethylation of cysteines was with 55 μM iodoacetamide for 30 min at room temperature (in the dark). Gel pieces were washed with gel wash buffer (25 mM ammonium bicarbonate and 50% acetonitrile) and dehydrated with 100% acetonitrile prior to drying in a SpeedVac vacuum centrifuge (Thermo). Trypsin digestion was carried out in 25 mM ammonium bicarbonate containing 10 μg mL⁻¹ MS-grade trypsin protease (Pierce), and the mixture was incubated overnight at 37°C. Peptides were subsequently extracted by a two-step process, which was repeated twice: incubation of gel pieces with 5% formic acid (Sigma-Aldrich), followed by 100% acetonitrile. To remove residual salts, the peptide samples were desalted using a HyperSep C₁₈ column (Thermo Fisher Scientific) according to a previously described procedure (84 (link), 85 (link)). C₁₈ column-eluted samples were dried by vacuum centrifugation and frozen prior to mass spectrometry.