Eppendorf 96 well plates (Microplate 96/V, wells clear, RecoverMax® well design, PCR clean, Eppendorf, Germany), were used for all preparations. Plates are washed with 50 % acetonitrile, 5-μl (microliter) in each well for 5 mins, then all liquid is shaken off, subsequently 1-μl of water is deposited by Mantis in each well in preparation for FACS (Fluorescence-activated cell sorting) sorting. After FACS sorting the cells in 3-nl of PBS buffer (Phosphate-Buffered Saline, pH 7.4, Thermo-Fisher, CA) the droplets go into 1-μl of 18 MΩ dd H2O, the cells are burst by mPOP method as previously described ( ), followed by 3 hours trypsin Gold™ (Promega, WI) digestion, TMT™ (Tandem Mass Tag, Thermo-Fisher, Germany) labeling and quenching procedures in a total volume of 1-μl . All procedures are completed in a fully automated manner by the Mantis robot. Buffers with single cell labeled peptides are first transferred into a glass vial (Thermo, CA) starting with 1μl of the carrier channel to passivate the glass surface and then all of single cell material is added to the vial. The final volume for MS injection is typically 10 microliters.