**4.3. Sample cleanup with C-18 ZipTips**

The resulting peptide digestion products were extracted by adding 100 μl of extraction buffer (1:2 (vol/vol) 5% formic acid/acetonitrile) to each tube and incubated for 15 min at 37 °C in a shaker. All extracts were saved and dried and re-dissolved in 10–20 μl of 0.1% FA, incubated for 2–5 min in the sonication bath and centrifuged for 15 min at 10,000 rpm at the bench-top centrifuge. The obtained supernatant was applied for binding the samples onto micro pipette tips (μC18), catalog number ZTC18S096 (ZipTip®, Millipore Co., Billerica, MA 01821 USA), equilibrated by being aspirated and dispensed with 100% acetonitrile, 40% acetonitrile/0.1% FA, 0.1% FA solutions. The samples were washed (4 times by aspirating and dispensing) with 15 μl of 0.1% FA), then eluted with 10 μl of 40% acetonitrile/0.1% FA. Appropriate aliquots were withdrawn for LC-MS/MS analysis or store at −20°C as contingency.
