Toward ‘Omic Scale Metabolite Profiling: A Dual Separation–Mass Spectrometry Approach for Coverage of Lipid and Central Carbon Metabolism

Abstract

Although the objective of any ‘omic science is broad measurement of its constituents, such coverage has been challenging in metabolomics because the metabolome is comprised of a chemically diverse set of small molecules with variable physical properties. While extensive studies have been performed to identify metabolite isolation and separation methods, these strategies introduce bias toward lipophilic or water-soluble metabolites depending on whether reversed-phase (RP) or hydrophilic interaction liquid chromatography (HILIC) is used respectively. Here we extend our consideration of metabolome isolation and separation procedures to integrate RPLC/MS and HILIC/MS profiling. An aminopropyl-based HILIC/MS method was optimized on the basis of mobile-phase additives and pH, followed by evaluation of reproducibility. When applied to the untargeted study of perturbed bacterial metabolomes, the HILIC method enabled the accurate assessment of key, dysregulated metabolites in central carbon pathways (e.g., amino acids, organic acids, phosphorylated sugars, energy currency metabolites), which could not be retained by RPLC. To demonstrate the value of the integrative approach, bacterial cells, human plasma, and cancer cells were analyzed by combined RPLC/HILIC separation coupled to ESI positive/negative MS detection. The combined approach resulted in the observation of metabolites associated with lipid and central carbon metabolism from a single biological extract, using 80 % organic solvent (ACN:MeOH:H2O 2:2:1). It enabled the detection of more than 30,000 features from each sample type, with the highest number of uniquely detected features by RPLC in ESI positive mode and by HILIC in ESI negative mode. Therefore, we conclude that when time and sample are limited, the maximum amount of biological information related to lipid and central carbon metabolism can be acquired by combining RPLC ESI positive and HILIC ESI negative mode analysis.

Ketogenesis prevents diet-induced fatty liver injury and hyperglycemia

“…Livers from control-and HMGCS2 ASO-treated mice were perfused with sodium [3-13 C]lactate (1.5 mM) and sodium [3-13 C]pyruvate (0.15 mM) as described above, then snap-frozen and pulverized. Four replicates of each sample type consisting of 10 to 20 mg of pulverized tissue were extracted with 1 ml of 2:2:1 acetonitrile (ACN)/methanol/water, using the protocol previously described by Ivanisevic et al (37). Briefly, samples were resuspended in solvent and subjected to repeated freeze-thaw cycles.…”

Assessment of altered lipid homeostasis by HILIC-ion mobility-mass spectrometry-based lipidomics

“…However, gradients for these separations tend to be long (30 min or longer), and sometimes two separate separations need to be performed to have complete coverage. LC separations by hydrophilic-interaction liquid chromatography (HILIC), which combines a hydrophilic stationary phase and reversephase mobile phase, were initially widely adopted for the analysis of small polar metabolites because of their increased retention on HILIC columns in relation to reversephase C18 columns (39)(40)(41). Recently, HILIC has been applied to the separation of phospholipid species as an alternative to lipid separations by normal-and reverse-phase chromatography (32,(42)(43)(44)(45).…”

Warpgroup: increased precision of metabolomic data processing by consensus integration bound analysis

“…To evaluate performance under a relevant set of conditions, we chose to generate one dataset with reversed-phase C18 chromatography and the second with amino propyl HILIC (Ivanisevic et al, 2013). Each dataset contained 11 LC/MS runs of Escherichia coli (E. coli) strain K12, MG1655 metabolic extract.…”

“…gas, 15; sheath gas, 30; counter gas, 0; capillary temperature, 310 C; sheath gas temperature, 200 C; spray voltage, 3.2 kV; needle diameter, 34 ga; s-lens, 65 V; mass range, 85-1165 Da; resolution 140 000; microscans, 1; max injection time; 200 ms; automatic gain control target: 3e6. HILIC was performed as described previously (Ivanisevic et al, 2013) by using the Phenomenex Luna NH2 (1.0 mm  150 mm  3 mm) column and a flow rate of 50 ml/min. Spectra were collected in negative ion mode.…”

“…Solvents were: A, 95% water þ 20 mM ammonium hydroxide þ 20 mM ammonium acetate; B, 100% acetonitrile. An injection volume of 1 ll was used with a linear gradient of (minutes, %A): 0, 5; 40, 100; 50, 100; 50.5, 40; 54.5, 15; 55, 5; 65, 5. Reversed-phase chromatography was performed as described previously (Ivanisevic et al, 2013) by using the Agilent Zorbax C18 (0.5 mm  150 mm  3 mm) column and a flow rate of 30 ml/min. Spectra were collected in positive ion mode.…”