Sera mag speedbeads

Samples were processed using an adapted SP3 protocol^{10 (link)}. Briefly, 200 μl reconstitution solution was added to each sample prepared in 50 μl NuPAGE LDS sample buffer (Life Technologies). Reduction was performed using 5 mM DTT with subsequent alkylation with 20 mM iodoacetamide. 10mM additional DTT was used for quenching. Equal volumes of two types of Sera-Mag Speed Beads (Cytiva, #45152101010250 and #65152105050250) were combined, washed with water and 10 μL of the bead mix were added to each sample. 260 μl 100% ethanol was added and samples were incubated for 5 min at 24 °C, 1000 rpm. Beads were captured on a magnetic rack for 2 min, and the supernatant was removed. Beads were washed twice with 200 μl 80% ethanol (Chromasolv, Sigma) and then once with 1000 μl 80% ethanol. Digestion was performed on beads with 0.25 μg Trypsin (Gold, Mass Spectrometry Grade, Promega) and 0.25 μg Lys-C (Wako) in 100 μl 100 mM ammonium bicarbonate at 37 °C overnight. Peptides were desalted using C-18 Stage Tips^{11 (link)}. Each Stage Tip was prepared with three discs of C-18 Empore SPE Discs (3 M) in a 200 μl pipet tip. Peptides were eluted with 60 % acetonitrile in 0.1 % formic acid, dried in a vacuum concentrator (Eppendorf), and stored at −20 °C. Peptides were dissolved in 2 % acetonitrile / 0.1 % formic acid prior to nanoLC-MS/MS analysis.