6490 triple quadrupole lc ms system

All samples were collected and treated following recently accepted guidelines for lipidomics analysis (79 (link)). Lipids were extracted from murine lung tissue, BALF, or isolated primary AEC2 cells from equal amounts of material (100 μg/sample) by a chloroform-methanol extraction method and lipid extracts analyzed as described in the Supplemental Methods using a 6490 Triple Quadrupole LC/MS system (Agilent Technologies) spiked with appropriate internal standards.

Lipids were extracted from equal amounts of material (30 μg protein/sample). Lipid extracts were prepared via chloroform–methanol extraction, spiked with appropriate internal standards, and analyzed using a 6490 Triple Quadrupole LC/MS system (Agilent Technologies, Santa Clara, CA) as described previously (Chan et al, 2012). Cholesterol and cholesteryl esters were separated with normal‐phase HPLC using an Agilent Zorbax Rx‐Sil column (inner diameter 2.1 Å ~100 mm) under the following conditions: mobile phase A (chloroform:methanol:1 M ammonium hydroxide, 89.9:10:0.1, v/v/v) and mobile phase B (chloroform:methanol:water:ammonium hydroxide, 55:39.9:5:0.1, v/v/v/v); 95% A for 2 min, linear gradient to 30% A over 18 min and held for 3 min, and linear gradient to 95% A over 2 min and held for 6 min. Quantification of lipid species was accomplished using multiple reaction monitoring (MRM) transitions that were developed in earlier studies (Chan et al, 2012) in conjunction with referencing of appropriate internal standards. Values are represented as mole fraction with respect to total lipid (% molarity). For this, lipid mass of any specific lipid is normalized by the total mass of all lipids measured (Chan et al, 2012).

Dried peptides were reconstituted in 3% acetonitrile, 97% water, and 0.1% formic acid (volume fraction). Separation and MRM analysis was performed on an Agilent Zorbax Eclipse Plus C18 RRHD column (2.1 mm x 50 mm, 1.8 μm particle) coupled to Agilent 6490 Triple Quadrupole LC/MS system with iFunnel technology (Santa Clara, CA). Peptides were eluted over 30 min gradient from 15% to 35% acetonitrile containing 0.1% (volume fraction) formic acid at a flow rate of 200 μL/min. Acquisition method used following parameters in positive mode: fragmentor 380 V, cell accelerator 4 V, electron multiplier 500 V, and capillary voltage 3500 V. Collision energy was optimized for each peptide using the default equation from Agilent, CE = 0.036 m/z—4.8 [29 (link)]. Dwell times were varied based on complexity of samples being analyzed and were in 80 ms to 120 ms range.

The analysis was performed by comparing the retention time and ionic characteristics of the HPLC–MS detection method. Standards of D2HG (H8378) and 3-methylhistidine (M9005) were purchased from Sigma-Aldrich LLC(USA). Tumor tissues (10 mg) were extracted by grinding with liquid nitrogen, and tumor fragments were homogenised in 1000 μL of pre-chilled 80% HPLC grade aqueous methanol. After centrifugation at 14,000 × g for 15 min, the supernatants were transferred to polypropylene tubes and evaporated under vacuum. The medium after culturing the IDH1 mutant and wild-type cancer cells for 24 h, 48 h and 72 h was collected, centrifuged, deproteinised and concentrated. The supernatants were also transferred to polypropylene tubes and evaporated under vacuum. Residues were reconstituted with 50 μL of methanol-water (90%:10%; v/v), and 10 μL was injected into an Agilent 6490 Triple Quadrupole LC/MS system. Serum D2HG and 3-methylhistidine levels were determined after comparison and calculation of the responsible peak area.

N2 and pld-1 animals were incubated in the presence or absence of ethanol (1%) for 1 h and all samples were immediately collected, frozen in liquid nitrogen and stored at −80 °C until further processing. Approximately 100 animals were used per sample. Lipids were subsequently extracted by a chloroform/methanol extraction, as previously described^{43 (link),44 (link)}. Lipid species were analyzed using a 6490 Triple Quadrupole LC/MS system (Agilent Technologies, Santa Clara, CA) operated in multiple reactions mode (MRM). PA and PEtOH levels were quantified by comparing to spiked internal standards diC17-PA and diC16-PEtOH (Avanti Polar Lipids). Lipid concentration was normalized by molar concentration across all species for each sample, and the final data is presented as the mean mol %^{43 (link),44 (link)}.

D2HG levels in sera were determined using a 6490 Triple Quadrupole LC/MS¹² . Briefly, 200 μL of thawed serum was mixed with 800 μL of acetonitrile and centrifuged at 10,000 rpm for 10 min at room temperature (24 °C). The supernatants were then transferred to polypropylene tubes and evaporated under a vacuum. The residue was reconstituted with 50 μL of 90% methanol in water (v/v), and 10 μL was injected into an Agilent 6490 Triple Quadrupole LC/MS system. Chromatographic separation was performed on a Chirex 3126 d-penicillamine, LC column (150 × 4.6 mm, Phenomenex, Torrance, CA, USA). Data acquisition, peak integration, and processing were performed using MassHunter software (Agilent Technologies, Santa Clara, CA, USA). MS-grade reagents were used for extractions. Pure D2HG was used as standard for the calculation of D2HG levels.