Spe buffer 3

The WCE if stored at -80°C was thawed to RT and then diluted with SPE buffer-1 (Thermo Fisher Scientific, proprietary) before SPE cleanup. Conditioning and equilibration of the Reverse Phase monolith (RP4H) SPE tips (Thermo Fisher Scientific, proprietary) was performed by placing them in a 96 well-plate with 50 µL SPE buffer-1 and SPE buffer-2 (Thermo Fisher Scientific, proprietary), respectively. The plates were then centrifuged at 2000 × g for 2 minutes at RT. Diluted WCE (50 µL) was loaded into the SPE tips and centrifuged. Washing of the tips was carried out with 50 µL of proprietary SPE buffer-3 (Thermo Fisher Scientific, proprietary). The tips were then placed in liquid chromatography (modified Easy-1000, Thermo Fisher Scientific) autosampler plate for further online elution and mass spectrometric (MS) analysis. For negative control samples tips designated as blanks were prepared in a similar way as described above, where instead of cell lysate water was added to the SPE tips.

The whole cell extract (WCE) was diluted with SPE buffer-1 (Thermo Fisher Scientific, proprietary) before Solid-Phase Extraction (SPE) cleanup. Reverse Phase monolith (RP4H) SPE tips (Thermo Fisher Scientific, proprietary) were placed in 96 well-plate and conditioned and equilibrated with 50 µL proprietary SPE buffer-2 (Thermo Fisher Scientific, proprietary), respectively. A centrifuge (Laboratory centrifuge 4-15C, Thermo Fisher, Osterode, Germany) capable of handling two 96 well-plates was used for centrifugation at 2000 × g for 2 minutes at RT. Diluted WCE (50 µL) was loaded into the SPE tips and centrifuged. The tips were washed with 50 µL of proprietary SPE buffer-3 and placed in liquid chromatography (modified Easy-1000, Thermo Fisher Scientific, proprietary) autosampler plate for further online elution and mass spectrometric analysis. Blanks were treated as negative controls and were prepared in a similar way as described above, instead of cell lysate water was added to the SPE tips.