2.5. Desalting of Tryptic Peptide Mixturesby Ziptip

Millipore Ziptips, micro-C18 (Sigma-Aldrich, #Z720003-96EA), were used for desalting the peptide mixtures. Other Ziptip kits such as C18 (Sigma-Aldrich, #Z720046-96EA) or C18 (ThermoFisher Scientific, Rockford, IL, USA #87782) can also be used in this protocol.

The samples were adjusted to 10 µL of 0.1% of TFA, at a final pH < 4. In order to wet the sorbent medium, 5 µL of acetonitrile was aspirated into a Ziptip by moving the pipettor plunger up and down 7–8 times. The solvent was discarded. For equilibration of the sorbent medium, 5 µL of 0.1% TFA was aspirated into the Ziptip by moving the pipettor plunger up and down 7–8 times. The solvent was discarded. For maximum binding of the peptides’ mixtures to thee Ziptip pipette tip, the samples were aspirated and dispensed 7–8 times. Then, 5 µL of 0.1% TFA was aspirated into the Ziptip, and the sorbent medium was washed by moving the pipettor plunger up and down 7–8 times. The solvent was discarded. At this point, 5 µL of 0.1% TFA/50% acetonitrile elution solution was aspirated into the Ziptip and dispensed into a clean Eppendorf tube. The solvent was removed in a vacuum concentrator, resuspended in 15 μL of solution containing 0.1% TFA for mass spectrometry experiments, and transferred to an HPLC vial. The dried peptides can be stored at −20 °C.