Spe buffer 2

The whole cell extract (WCE) was diluted with SPE buffer-1 (Thermo Fisher Scientific, proprietary) before SPE (solid-phase extraction) cleanup. RP4H SPE tips (Thermo Fisher Scientific, proprietary) were placed in a 96-well plate and conditioned and equilibrated with 50 µL proprietary SPE buffer-2 (Thermo Fisher Scientific, proprietary), respectively. Centrifuge (Laboratory centrifuge 4-15C, Thermo Fisher Scientific, Osterode, Germany) capable of handling two 96-well plates was used for centrifugation at 2000× g for 2 min at room temperature. Diluted WCE (50 µL) was loaded into the SPE tips and centrifuged. The tips were washed with 50 µL of proprietary SPE washing buffer and placed in Stand-Alone Liquid Chromatography (SALC) autosampler plate for further online elution and mass spectrometric (MS) analysis.

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.