Comprehensive Profiling of Metabolites and Lipids in Plasma

Profiles of endogenous, polar metabolites and lipids were obtained using LC-MS. The polar metabolite profiling methods were developed using reference standards of each metabolite to determine chromatographic retention times and MS multiple reaction monitoring transitions, declustering potentials and collision energies (See Supplemental Data Tables 1-3 for LC-MS parameters for each method).
Negative ionization mode data were acquired using an ACQUITY UPLC (Waters) coupled to a 5500 QTRAP triple quadrupole mass spectrometer (AB SCIEX) running a modified version of the hydrophilic interaction chromatography (HILIC) method described by Bajad et al. (20 (link)). Plasma samples (30 μL) were extracted using 120 μL of 80% methanol (VWR) containing 0.05 ng/μL inosine-¹⁵N₄, 0.05 ng/μL thymine-d₄, and 0.1 ng/μL glycocholate-d₄ as internal standards (Cambridge Isotope Laboratories). The samples were centrifuged (10 min, 9,000 × g, 4°C) and the supernatants (10 μL) were injected directly onto a 150 × 2.0 mm Luna NH2 column (Phenomenex) that was eluted at a flow rate of 400 μL/min with initial conditions of 10% mobile phase A (20 mM ammonium acetate and 20 mM ammonium hydroxide (Sigma-Aldrich) in water (VWR)) and 90% mobile phase B (10 mM ammonium hydroxide in 75:25 v/v acetonitrile/methanol (VWR)) followed by a 10 min linear gradient to 100% mobile phase A. The ion spray voltage was −4.5 kV and the source temperature was 500°C.
Positive ionization mode data were acquired as in Wang et al. with a modification to the MS acquisition in which all multiple reaction monitoring transitions were scheduled in a single method file (3 (link)). Briefly, the LC-MS system consisted of a 4000 QTRAP triple quadrupole mass spectrometer (AB SCIEX) coupled to an 1100 Series pump (Agilent) and an HTS PAL autosampler (Leap Technologies). Plasma samples (10 μL) were extracted using nine volumes of 74.9:24.9:0.2 (v/v/v) acetonitrile/methanol/formic acid containing stable isotope-labeled internal standards (0.2 ng/μL valine-d8, Isotec; and 0.2 ng/μL phenylalanine-d8 (Cambridge Isotope Laboratories)). The samples were centrifuged (10 min, 9,000 × g, 4°C) and the supernatants (10 μL) were injected onto a 150 × 2.1 mm Atlantis HILIC column (Waters). The column was eluted isocratically at a flow rate of 250 μL/min with 5% mobile phase A (10 mM ammonium formate and 0.1% formic acid in water) for 1 minute followed by a linear gradient to 40% mobile phase B (acetonitrile with 0.1% formic acid) over 10 minutes. The ion spray voltage was 4.5 kV and the source temperature was 450°C.
Lipids were profiled as described in Rhee et al. (21 (link)). Briefly, plasma samples (10 μL) were extracted for lipid analyses with 190 μL of isopropanol containing 0.25 ng/μL 1-dodecanoyl-2-tridecanoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids). After centrifugation, supernatants (10 μL) were injected directly onto a 150 × 3.0 mm Prosphere HP C4 column (Grace). The column was eluted isocratically with 80% mobile phase A (95:5:0.1 vol/vol/vol 10mM ammonium acetate/methanol/acetic acid) for 2 minutes followed by a linear gradient to 80% mobile-phase B (99.9:0.1 vol/vol methanol/acetic acid) over 1 minute, a linear gradient to 100% mobile phase B over 12 minutes, then 10 minutes at 100% mobile-phase B. MS analyses were carried out using electrospray ionization and Q1 scans in the positive ion mode. Ion spray voltage was 5.0 kV and source temperature was 400°C.
Prior to each set of analyses, LC-MS system sensitivity and chromatography quality were checked by analyzing reference samples: synthetic mixtures of reference metabolites (Sigma) and a lipid extract prepared from a pooled human plasma stock (Bioreclamation). During the application of each method, internal standard peak areas were monitored for quality control. Moreover, reference pooled plasma samples (which were not blinded to the laboratory, unlike the QC pooled plasma samples in the blinded duplicates pilot) were included in each set of analyses, with samples inserted at the beginning and after sets of approximately twenty study samples. The reproducibility of each metabolite in the pooled plasma samples was determined to confirm the overall quality of the analyses (See Supplemental Data Tables 4-6). MultiQuant 1.2 software (AB SCIEX) was used for automated peak integration and metabolite peaks were manually reviewed for quality of integration and compared against a known standard to confirm identity. Metabolites with a signal to noise ratio <10 were considered unquantifiable. For these analyses, metabolite signals were retained as measured LC-MS peak areas, which are proportional to metabolite concentration and are appropriate for metabolite clustering and correlative analyses.