6550 ifunnel q tof lc ms instrument

IM and OM lipids of C. glutamicum were extracted and analysed as previously described [27 (link)]. In brief, four replicates of WT C. glutamicum and mutant strain were grown to the exponential phase (OD_600nm = 2.5–3) in BHI medium and the cells were harvested by centrifugation. The cell pellet was extracted with water-saturated 1-butanol to selectively remove OM lipids. After another centrifugation step, the same pellet was sequentially extracted in chloroform:methanol (2:1, v/v) and chloroform:methanol:water (1:2:0.8 v/v) to extract the remaining lipids of the IM. Both IM and OM lipids were separated on an Agilent 1290 Infinity Quaternary LC System (Agilent Technologies) using a C18 column (Phenomenex Kinetex, 2.6 um EVO C18 100A) eluted with an isopropanol mobile phase binary solvent system at a flow rate of 260 µL/min. Eluted lipids were analysed on a 6550 iFunnel Q-TOF LC/MS instrument (Agilent Technologies) in positive ionization mode. Lipids were identified based on their mass-to-charge ratio and fragmentation pattern using a lipid library [27 (link)]. Statistical analyses were performed using MetaboAnalyst.

All starting materials and chemicals were supplied by Alfa Aesar and Sigma Aldrich. Solvents were purchased from Bio-Lab Israel.
Chemical reactions were monitored by TLC (Silica gel 60 F-254, Merck) and LCMS. LCMS analyses were performed using an Agilent Technologies 1260 Infinity (LC) 6120 quadruple (MS) instrument, column Agilent SB-C18, 1.8 mm, 2.1 × 50 mm, column temperature 50 °C, eluent water–acetonitrile (ACN) + 0.1% formic acid. HRMS analyses were performed in ESI positive mode by using an Agilent 6550 iFunnel Q-TOF LCMS instrument. ¹H NMR and ¹³C NMR spectra were measured in DMSO-d_6, CD₃OD, and a mixture CDCl₃–CD₃OD on a Bruker Avance III HD (400 MHz and 100 MHz for ¹H and ¹³C, respectively) spectrometer. Chemical shifts were reported in ppm using the solvent residual signal as an internal reference: δ_H(DMSO-d₆) = 2.50 ppm, δ_C(DMSO-d₆) = 39.52 ppm; δ_H(CDCl₃) = 7.26 ppm, δ_C(CDCl₃) = 77.16 ppm; δ_H(CD₃OD) = 3.31 ppm, δ_C(CD₃OD) = 49.0 ppm.