Speedvac vacuum concentrator

As described previously, the labelled peptide mixtures were fractionated using the 3100 OFFGEL fractionator kit (Agilent Technologies) on 12-cm immobilized pH gradient strips (IPG; pH 3–10)^{92 (link)}. Rehydration and running buffer contained 0.1% IPG ampholyte solution and 0.3% glycerol. Peptides were focused using 50 μA current and 200 mW power to achieve a final voltage of 50 kV h. Peptide fractions were then incubated with 50 μl of a solution containing methanol, water, and trifluoroacetic acid at a ratio of 50:49:1 for 15 min prior to centrifugation in a SpeedVac vacuum concentrator (Eppendorf) and storage at − 80 °C. To solubilize the peptides, a solution containing 1% acetonitrile and 0.05% trifluoroacetic acid was used prior to desalting using C₁₈ StageTips^{93 (link)}. To pack the StageTips, 3 C₁₈ disks were used and incubated with methanol for activation. Prior to loading, peptides were washed 2 times using a solution containing 2% acetonitrile and 0.1% trifluoroacetic acid. A solution containing 0.1% acetic acid and 2% acetonitrile and 0.1% trifluoroacetic acid was used to wash the StageTips prior to an elution step with 60% acetonitrile and 0.1% trifluoroacetic acid. Samples were dried by centrifugation in a SpeedVac vacuum concentrator and resuspended in mobile phase A (0.5% acetic acid) before mass-spectrometric analysis.

Tissue samples were homogenised with 50 μL of urea (8 M) and incubated for 1.5 h at room temperature. Samples were subsequently centrifuged at 2000 g for 5 minutes, and the supernatant was quantified using the Qubit Protein Assay kit (Life Technologies, Waltan, MA, US). We used 30 μg of protein for each sample, which was reduced with 5 mM DTT (1,4-dithiothreitol, Sigma-Aldrich, St. Louis, MO, US) at 56 °C for 25 minutes and alkylated with 14 mM iodoacetamide (Sigma-Aldrich, St. Louis, MO, US) at room temperature for 30 minutes in the dark. After the addition of 1 mM CaCl₂ (final concentration), proteins were digested with 0.6 μg of trypsin (Promega, Madison, WI, US sequence grade) by incubation for 16 h at room temperature. The reaction was stopped by adding TFA (trifluoroacetic acid, Sigma-Aldrich, St. Louis, MO, US) to a final concentration of 0.4%. Samples were centrifuged at 2500 g for 10 minutes at room temperature, and proteins were desalted using SepPack columns (Waters, C18) with acetonitrile (MS-grade acetonitrile from Sigma-Aldrich, St. Louis, MO, US), dried in SpeedVac vacuum concentrator (Eppendorf), and stored at −20 °C.