Model 5301

All samples (n = 6) were diluted in 100 μL of 50 mM ammonium bicarbonate buffer pH 8.5 with 0.01% ProteaseMax (Promega, Madison, WI, USA) and 20 mM DTT, and incubated for 20 min at 56 °C. After this process, the samples were blocked by adding 100 mM iodoacetamide and incubated for 30 min at room temperature in the dark. Finally, the samples were digested by adding 1 μg trypsin (Trypsin Gold Mass Spectrometry Grade (V5280), Promega, Madison, WI, USA) for 3 h at 37 °C. The reaction was stopped with 0.1% formic acid and filtered through a 0.2 μm pore filter. The samples were dried using a vacuum concentrator (Model 5301, Eppendorf, Hamburg, Germany).

Solid-phase extraction (SPE) was conducted using Chromabond Multi 96-well plate (Macherey–Nagel, Düren, Germany) containing 50 mg/well HR-X-resin (Macherey–Nagel, Düren, Germany). First, the SPE plate was conditioned by 1 mL of methanol followed by 1 mL of water. In this and all subsequent steps, the liquid was passed through the resin by centrifugation at 500×g for 5 min using JS5.3 swingout rotor in an Avanti J-26XP centrifuge (Beckman Coulter, Fullerton, CA, USA). 500 µL of 5% (v/v) acetonitrile was added to the sample and standard solutions mentioned in “Sample derivatization and processing”. Then, the resulting solutions were quantitatively loaded onto the SPE plate, washed with 1 mL of water and the flow through was discarded after centrifugation. In the next step, 1 mL of methanol was added into the 96-deep well plate and eluted to a new block by centrifugation. Finally, the eluates were transferred from the 96-deep well block to 2 mL Eppendorf tubes, and allowed to evaporate under vacuum in an Eppendorf Concentrator (Model 5301, Eppendorf, Hamburg, Germany) at 45 °C for 45 min. Finally, the samples were centrifuged (Model 5415C, Eppendorf^®, Germany) at 10,000×g for 10 min and the supernatant was transferred to the 96-well plate and the plate was placed in LC–MS/MS auto-sampler.