Gemini guard column

Rv, RvΔembR, and RvΔembR::embR strains were grown in 7H9-ADC either under regular growth conditions or subjected to hypoxic stress. Cells equivalent to an OD₆₀₀ of ~6 were washed thrice with cold, sterile PBS and resuspended in 1 mL PBS. The resuspended cells were transferred into a glass vial. The cellular lipids were extracted using an established protocol (33 (link)) by adding 3 mL of 2:1 (vol/vol) chloroform-methanol mixture. Samples were vortexed and centrifuged at 2,800 × g (15 min at 4°C), and the organic layer was collected. The remaining aqueous layer was acidified (2% formic acid) and reextracted using chloroform (2 mL) to enrich phospholipids. The samples were once again centrifuged, and the organic layer was collected. The organic layers were pooled and dried under a stream of nitrogen gas at room temperature. The dried pellets were resuspended 200 μL of 2:1 (vol/vol) chloroform-methanol and subjected to semiquantitative (relative quantification) information-dependent acquisition (IDA)-mediated LC-MS/MS analysis (34 (link), 35 (link)) on a Sciex X500R quadrupole time-of-flight (QTOF) mass spectrometer fitted with an Exion ultrahigh-performance LC system. The LC was performed on a Gemini 5U C₁₈ column (5 μm, 50 by 4.6 mm; Phenomenex) coupled to a Gemini guard column (4 by 3 mm; Phenomenex security cartridge; Phenomenex).

Cultures were harvested and suspended in an appropriate volume of 100 mM Tris-Cl (pH 8.0) and disrupted. The resultant whole-cell lysate was acidified by addition of 6 M HCl. Low-molecular-weight molecules were then extracted by adding a double volume of ethyl acetate and left overnight on a stirrer. The organic layer was then separated and evaporated to dryness, and the residual material was dissolved in a minimal volume of methanol and analyzed by information-dependent acquisition (IDA) scanning on a Sciex X500R quadrupole time of flight (QTOF) mass spectrometer fitted with an ExionLC UPHLC system using Sciex OS software with a previously reported method (45 (link), 46 (link)). The LC separation was achieved on a Gemini 5U C₁₈ column (Phenomenex; 5 μm, 50 by 4.6 mm) coupled to a Gemini guard column (Phenomenex; 4 by 3 mm, Phenomenex security cartridge). All metabolites, I to IX, were analyzed by iminodiacetic acid (IDA) scanning in both positive- and negative-ionization mode using an electrospray ionization (ESI) source with solvent systems, flow rates, and a solvent gradient described earlier (45 (link), 46 (link)). The total scan time for both the MS1 and MS2 spectra was 2.5 s, and a collision energy (volts) of 5 was used. The declustering potential and ion source voltage were set at 100 and 5,500 V respectively.