title: An LC-MS-based chemical and analytical method for targeted metabolite quantification in the model cyanobacterium Synechococcus sp. PCC 7002.
abstract: Herein, we detail the development of a method for the chemical isolation and tandem LC-MS/MS quantification of a targeted subset of internal metabolites from cyanobacteria. We illustrate the selection of target compounds; requirements for and optimization of mass spectral detection channels, screening, and optimization of chromatography; and development of a sampling protocol that seeks to achieve complete, representative, and stable metabolite extraction on the seconds time scale. Several key factors influencing the separation by reversed-phase ion pairing chromatography, specifically the hydrophobicity of the sample matrix and sensitivity to mobile phase acidity, are identified and resolved. We illustrate this methodology with an example from the autofermentative metabolism in the model cyanobacterium Synechococcus sp. PCC 7002, for which intracellular levels of 25 metabolites were monitored over 48 h, including intermediates in central carbon metabolism together with those involved in the cellular energy charge and redox poise. Upon removal of alternative reductant sinks (nitrate), anoxia induces autofermentation of carbohydrates with a parallel rise in the intracellular pyridine nucleotide redox poise that is specific to NAD(H) and alongside a gradual decline in the adenylate energy charge. This LC-MS/MS-based method provides for accurate time-resolved quantification of multiple metabolites in parallel, thus enabling experimental verification of the active metabolic pathways.
authors.affiliation: Waksman Institute and Department of Chemistry & Chemical Biology, Rutgers The State University of New Jersey, 190 Frelinghuysen Road, Piscataway, New Jersey 08854, USA.
authors.forename: Nicholas B
authors.initials: NB
authors.lastname: Bennette
authors.forename: John F
authors.initials: JF
authors.lastname: Eng
authors.forename: G Charles
authors.initials: GC
authors.lastname: Dismukes
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D053858:Metabolic Networks and Pathways; D055442:Metabolome; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D011742:Pyrimidine Nucleotides / Q000737:chemistry / Q000378:metabolism; D046940:Synechococcus / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D011742:Pyrimidine Nucleotides; D009243:NAD