Thermo q exactive orbitrap

Intact polar lipids (IPLs) were extracted from S. denitrificans, grown at different NaCl concentrations (0, 10, 20 g/L), using a modified Bligh and Dyer extraction (Bligh & Dyer, 1959; Popendorf, Fredricks, & Van Mooy, 2013) with 2,4‐dinitrophenyl modified phosphatidylethanolamine diacylglycerol (DNP‐PE‐C_16:0/C_16:0‐DAG; Avanti Polar Lipids Inc., Alabaster, AL) as internal standard. IPLs were analyzed by normal phase HPLC‐MS on an Agilent 1200 HPLC (Agilent, Santa Clara, CA, USA) coupled to a Thermo Q Exactive Orbitrap high‐resolution mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA). Chromatography was as described by Popendorf et al. (2013) and mass spectrometry conditions were modified after Collins, Edwards, Fredricks, and Van Mooy (2016). Lipid abundances were corrected for response factors of adequate standards. The abundances of IPLs with phosphatidylglycerol (PG) and PE head groups were corrected by the relative responses of commercial DAG‐C_16:0/C_16:0 standards with the respective head group (Avanti Polar Lipids, Inc., Alabaster, AL, USA). Individual PG‐ and PE‐DAG lipids were identified by the total number of acyl carbons and double bonds (C_n:m, where n indicates the number of carbon atoms and m the number of double bonds). The detailed molecular structure of fatty acids cannot be identified by positive ion HPLC‐MS.

The instrumental analyses were performed by a Dionex ICS-5000+ Ionic Chromatography (IC) system (Sunnyvale, CA, USA) coupled to a Thermo Q-Exactive Orbitrap™ (Thermo Scientific, San Jose, CA, USA), equipped with a heated electrospray ionization (HESI) source. For the analyte separation, a Thermo Scientific Dionex IonPac AS19-4 μm (2 × 250 mm, 4 μm particle size) with a Dionex IonPac AG19-4 μm guard column (2 × 50 mm) was used. The gradient, all IC, and HRMS parameters were described in Chiesa et al. [7 (link)].
Chromeleon^TM (Thermo Fisher Scientific, San Jose, CA, USA) and Xcalibur^TM 3.0 (Thermo Fisher Scientific, Waltham, MA) software was used to control the IC and HRMS system, respectively.

Ten microliters of each sample were injected in a constrained randomized order into a Thermo Ultimate 3000 HPLC equipped with a Thermo Accucore aQ RP C18 column (100 × 2.1 mm, 2.6 µm particle size) and coupled to a Thermo Q-Exactive Orbitrap (all purchased from Thermo Fisher Scientific, Hägersten, Sweden). The mass spectrometer was operated in positive and negative ion mode and MS resolutions were set to 70,000 at m/z 200, AGC target 1,000,000, and maximum ion injection time 250 ms. A QC and a blank injection were done every eighth sample. Finally, a 2-fold serial dilution series ranging from 0.5 to 32.0 µL QC was injected. For improving metabolite identification, eight tandem mass spectrometry analyses in both ion modes were performed separately on pooled samples stratified on the diagnostic groups.