An LC–MS-Based Chemical and Analytical Method for Targeted Metabolite Quantification in the Model Cyanobacterium Synechococcus sp. PCC 7002

摘要

ABSTRACT: Herein, we detail the development of a methodnfor the chemical isolation and tandem LCu0001MS/MS quantifica-ntion of a targeted subset of internal metabolites from cyano-nbacteria. We illustrate the selection of target compounds;nrequirements for and optimization of mass spectral detectionnchannels, screening, and optimization of chromatography; andndevelopment of a sampling protocol that seeks to acheievencomplete, representative, and stable metabolite extraction onnthe seconds time scale. Several key factors influencing the separation by reversed-phase ion pairing chromatography, specifically thenhydrophobicity of the sample matrix and sensitivity to mobile phase acidity, are identified and resolved. We illustrate thisnmethodology with an example fromthe autofermentativemetabolismin themodel cyanobacteriumSynechococcus sp. PCC 7002, fornwhich intracellular levels of 25metabolites weremonitored over 48 h, including intermediates in central carbonmetabolismtogethernwith those involved in the cellular energy charge and redox poise. Upon removal of alternative reductant sinks (nitrate), anoxianinduces autofermentation of carbohydrates with a parallel rise in the intracellular pyridine nucleotide redox poise that is specifictonNAD(H) and alongside a gradual decline in the adenylate energy charge. This LCu0001MS/MS-based method provides for accuratentime-resolved quantification of multiple metabolites in parallel, thus enabling experimental verification of the active metabolicnpathways.