Proteolytic digestion
Samples were processed by single-pot solid-phase-enhanced sample preparation (SP3) as described before (). In brief, proteins bound to the streptavidin beads were released by incubating the samples for 5 min at 95° in an SDS-containing buffer (1% (w/v) SDS, 5 mM biotin in water/Tris, pH 8.0). After elution, proteins were reduced and alkylated, using DTT and iodoacetamide (IAA), respectively. Afterwards, 2 µl of carboxylate-modified paramagnetic beads (Sera-Mag Speed Beads, GE Healthcare; Chicago, US; 0.5 μg solids/μl in water as described by ) were added to the samples. After adding acetonitrile to a final concentration of 70% (v/v), samples were allowed to settle at RT for 20 min. Subsequently, beads were washed twice with 70% (v/v) ethanol in water and once with acetonitrile. The beads were resuspended in 50 mM NH4HCO3 supplemented with trypsin (Mass Spectrometry Grade, Promega; Madison, US) at an enzyme-to-protein ratio of 1:25 (w/w) and incubated overnight at 37°C. After overnight digestion, acetonitrile was added to the samples to reach a final concentration of 95% (v/v) followed by incubation at RT for 20 min. To increase the yield, supernatants derived from this initial peptide-binding step were additionally subjected to the SP3 peptide purification procedure (). Each sample was washed with acetonitrile. To recover bound peptides, paramagnetic beads from the original sample and corresponding supernatants were pooled in 2% (v/v) dimethyl sulfoxide (DMSO) in water and sonicated for 1 min. After 2 min of centrifugation at 14,000xg and 4°C, supernatants containing tryptic peptides were transferred into a glass vial for MS analysis and acidified with 0.1% (v/v) formic acid.