Shrimp metabolomics

Metabolomics is a systemic approach to studying metabolic activity within the cells of a living organism. Liquid chromatography - electrospray – mass spectrometry (LC-ESI-MS) is used to measure the abundance of intracellular metabolites at nano molar sensitivities. Metabolomic techniques allow observation of the actual physiological phenomena that occur during virus-host interactions in vivo.

Extraction of metabolites

To extract the metabolites from shrimp hemocytes, hemolymph is collected using a syringe containing cooled anticoagulant solution (2 % NaCl, 0.1 M glucose, 30 mM sodium citrate, 26 mM citric acid, 10 mM EDTA). After centrifugation at 3000 x g for 10 min, the resulting hemocyte pellets are then rinsed with cold 1 x PBS and resuspended with 100μl 1 x PBS. This suspension may be stored at -80°C until use. The total metabolites are extracted from the suspension using 100 % MeOH (hemocytes suspension [μl]: buffer [μl] 1:2). The extracted hemolysate is centrifuge at 12000 x g for 10 min, and the supernatant is dried using a centrifugal evaporator (CVE-2000, EYELA). The pellet is suspended with 50μl ddH₂O for LC-ESI-MS analysis.

Liquid chromatography - electrospray - mass spectrometry

The LC-ESI-MS system consists of an ultra-performance liquid chromatography (UPLC) system (Ultimate 3000 RSLC, Dionex) and an electrospray ionization (ESI) source of quadrupole time-of-flight (TOF) mass spectrometer (maXis HUR-QToF system, Bruker Daltonics). With the autosampler set at 4 ℃, separation is performed using reversed-phase liquid chromatography (RPLC) on a BEH C18 column (2.1 x 100 mm, Walters). The elution starts with 1% mobile phase A (0.1% formic acid in deionized water) and 99% mobile phase B (0.1% formic acid in ACN), is held at 1% B for 0.5 min, raised to 60% B over 5 min, further raised to 90% B in 0.5 min, held at 90% B for 1.5 min, and then lowered to 1% B in 0.5 min. The column is equilibrated by pumping 1% B for 4 min. The flow rate is set to 0.4 ml/min with an injection volume of 10 μl. LC-ESI-MS chromatograms are acquired in negative ion mode under the following conditions: capillary voltage of 3500 V, dry temperature at 190 ℃, dry gas flow maintained at 8 l/min, nebulizer gas at 1.4 bar, and acquisition range of m/z 100-1000.