Evaluation of quenching methods for metabolite recovery in photoautotrophic Synechococcus sp. PCC 7002

Abstract

The first step of many metabolomics studies is quenching, a technique vital for rapidly halting metabolism and ensuring that the metabolite profile remains unchanging during sample processing. The most widely used approach is to plunge the sample into prechilled cold methanol; however, this led to significant metabolite loss in Synecheococcus sp. PCC 7002. Here we describe our analysis of the impacts of cold methanol quenching on the model marine cyanobacterium Synechococcus sp. PCC 7002, as well as our brief investigation of alternative quenching methods. We tested several methods including cold methanol, cold saline, and two filtration approaches.

Characterizing Photosynthetic Biofuel Production: Isotopically non-stationary13C metabolic flux analysis (INST-13CMFA) on limonene producingSynechococcussp. PCC 7002

“…Intracellular metabolites were extracted from the frozen cell pellets using a methanol extraction (Sake et al, 2020). Cell pellets were resuspended in 500 μL pure methanol and spiked with ribitol and PIPES internal standards for final concentrations of 150 ppb and 50 ppb, respectively.…”

“…The cultures were first grown up to mid-exponential phase (OD 730 ~0.8) at the conditions previously described. Before the addition of label, 15 mL of culture was pipetted out and immediately quenched in 30 mL of partially frozen saline quench solution in a 50 mL conical centrifuge tube, as described in Sake et al (Sake et al, 2020). This sample corresponded to the zero timepoint sample (t=0).…”

Chlamydomonas Mutants Null for Chloroplast Triose Phosphate Transporter3 are Metabolically Compromised and Light Sensitive

“…45 mL of culture was rapidly quenched in cold saline solution (-2 to -3°C), extracted using cold methanol and then analyzed by LC-MS/MS. Quenching and analysis of metabolites were performed according to (Sake et al, 2020) with modifications. The quenching solution, filtered saline (9 g/L NaCl), was prechilled to 4 o C in a refrigerator.…”

Characterizing Photosynthetic Biofuel Production: Isotopically Non-Stationary 13C Metabolic Flux Analysis on Limonene Producing Synechococcus sp. PCC 7002

“…The cultures were first grown to midexponential phase (OD 750 ~0.8) at the conditions previously described. Before the addition of the label, 15 ml of culture was pipetted out and immediately quenched in 30 ml of partially frozen saline quench solution in a 50 ml conical centrifuge tube, as described in Sake et al (Sake et al, 2020). This sample corresponded to the zero timepoint sample (t = 0).…”

“…Intracellular metabolites were extracted from the frozen cell pellets using a methanol extraction (Sake et al, 2020). Cell pellets were resuspended in 500 μl pure methanol and spiked with ribitol and PIPES internal standards for final concentrations of 150 and 50 ppb, respectively.…”