Discovery dsc 18 tube

Сells were washed three times with PBS, pH 7.5. Cell pellet was treated with 3 μl of 10% RapiGest SF (Waters) and 1 μl nuclease mix for 30 min at 4 °C, then resuspended in 37 μl of 100 mM NH₄HCO₃, vortexed and heated at 100 °C for 5 min. After cooling to room temperature cell debris was removed by centrifugation at 15,000 g for 5 min. Protein cysteine bonds were reduced with 10 mM DTT in 5 mM NH₄HCO₃ for 30 min at 60 °C and alkylated with 30 mM iodoacetamide in the dark at RT for 30 min. The step with adding DTT was repeated. Clarified extract protein concentration was estimated using Bradford Protein Assay Kit (BioRad). Trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega) was added in 1/50 w/w trypsin/protein ratio and incubated at 37 °C overnight. To stop trypsinolysis and degrade the acid-labile RapiGest surfactant, trifluoroacetic acid (TFA) was added to the final concentration of 0,5% v/v (the pH should be less than 2.0), incubated at 37 °C for 45 min and the samples were centrifugated at 15,000 g for 10 min to remove the surfactant. Hydrolyzate was desalted using a Discovery DSC-18 Tube (Supelco) according to the manufacturer protocol. Peptides were eluted with 700 μL 75% ACN, 0.1% TFA, dried in a SpeedVac (Labconco) and resuspended in 3% ACN, 0.1% TFA to the final concentration of 5 μg/μL.

The nitrocellulose filter fragments were covered with 100 mm NH₄HCO₃ containing 0.1% sodium deoxycholate (DCNa), incubated in an ultrasonication bath for 10 min and heated for 5 min at 95 °C. After cooling the samples, 5 mm of the reducing agent tris(2‐carboxyethyl)phosphine (TCEP) was added and the samples were incubated at 37 °C for 1 h. Then, 30 mm iodoacetamide (IAA) was added and the samples were maintained at room temperature (RT) for 30 min. To avoid chemical modifications and remove the unreacted IAA, the samples were treated with 2.5 mm TCEP and incubated at RT for 30 min. Protein hydrolysis was performed with trypsin (20 µg per sample, Trypsin Gold, Mass Spectrometry Grade; Promega) for 16 h at 37 °C. After that, an equal volume of ethyl acetate was added to the sample and its pH was adjusted to 2.0 with trichloroacetic acid (TCA). At this point, trypsinolysis stopped and DCNa was precipitated and moved to the organic phase. After 5 min of intensive shaking and 5 min of centrifugation at 16 000 g at 20 °C, the lower water phase was collected and cleaned with C18 cartridges [Discovery DSC‐18 Tube (Supelco; Sigma‐Aldrich)] according to the manufacturer's protocol. The achieved peptide extracts were dried in a SpeedVac (Labconco, Kansas City, MO, USA) and dissolved in 15 µL of HPLC‐MS/MS sample buffer containing 3% acetonitrile and 0.1% trifluoroacetic acid.