4. Metabolomics

1. Context

Metabolomics pemits the quantitative sampling of the entire suite of detectable metabolites in a biological system, and thus provides a holistic view of physiological processes (Malmendal et al. 2006, Michaud & Denlinger 2007). The advantage of metabolomics for analyzing changes in a biological context is the unbiased nature of the analysis; all detectable metabolites are quantified to eliminate a priori determination of analytical targets. Classical metabolomics allows the identification of unexpected correlations between apparently unrelated pathways of metabolism (Fiehn, 2002). Also, metabolomics is the only field of “–omics” that addresses the terminal target of the central dogma of molecular biology, the substrate. Metabolomics promises to provide useful insights for insect physiology. The equipment and analytical procedures are being routinely used in our lab.

2. Our Methodological procedure

2.1. Sample preparation and derivatization

The samples are homogenized in ice-cold (-20 °C) methanol-chloroform solution (2:1) using a tungsten-bead beating apparatus (RetschTM MM301, Retsch GmbH, Haan, Germany) at 25 agitations per second for 1.5 min. Then, ice-cold ultrapure water is added to the samples that are subsequently vortexed. After centrifugation, an aliquot of the upper aqueous phase (which contained polar metabolites) is transferred to new chromatographic glass vials. The vials containing the aliquots are vacuum-dried using a Speed Vac Concentrator (MiVac, Genevac Ltd., Ipswitch, England). The dried aliquots are resuspended in of 20 mg. L-1 methoxyamine hydrochloride in pyridine, incubated under automatic orbital shaking. Then, 30 µl of N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) is added and the derivatization is conducted under agitation.

All the derivatization process is automatized using CTC CombiPal autosampler (GERSTEL GmbH and Co.KG, Mülheim an der Ruhr, Germany). Our innovative and uncommon analytical procedure ensures identical derivatization time and process for all samples, and represents a prerequisite for a total quality control.

2.2. GC-MS analyses

Our GC-MS system consists of a Trace GC Ultra chromatograph and a Trace DSQII quadrupole mass spectrometer (Thermo Fischer Scientific Inc, Waltham, MA, USA, Figure 1). The injector temperature is held at 250 °C and we are using a 30 m fused silica column (TR5 MS, I.D. 25 mm, 95% dimethyl siloxane, 5% Phenyl Polysilphenylene-siloxane) with helium gas as the carrier at a rate of 1 ml.min-1. One microliter of each sample is injected using the splitless mode (25:1), and detection is achieved using MS detection in electron impact. We completely randomize the injection order of the samples. Most often, we use the selective ion monitoring mode (SIM) (electron energy: -70 eV), ensuring a precise annotation of the detected peaks. Then, we search for the metabolites that were included in our spectral database (63 pure reference compounds). The peaks are accurately annotated using both their mass spectra (two specific ions) and their retention time. Calibration curves were set using samples consisting of 60 pure reference compounds at levels of 1, 2, 5, 10, 20, 50, 100, 200, 500, 750, 1000, 1500 and 2000 µM. Chromatograms are deconvoluted using XCalibur v2.0.7 software (Thermo Fischer Scientific Inc, Waltham, MA, USA). Metabolite levels were quantified according to their calibration curves.

Figure 1: Picture of our GC-MS system

References:

Fiehn O. (2002) Metabolomics - the link between genotypes and phenotypes. Plant Mol Biol 48:155–171.

Malmendal A., Overgaard J., Bundy J.G., et al. (2006) Metabolomic profiling of heat stress: hardening and recovery of homeostasis in Drosophila. Am J Physiol Regulatory Integrative Comp Physiol 291(1):205-212.

Michaud M.R. & Denlinger D.L. (2007) Shifts in the carbohydrate, polyol, and amino acid pools during rapid cold-hardening and diapause-associated cold-hardening in Xesh Xies (Sarcophaga crassipalpis): a metabolomic comparison. J Comp Physiol B 177:753-763.