Sensitive characterization of microbial ubiquinones from biofilms by electrospray/mass spectrometry

Utilizing high-performance liquid chromatography/electrospray/tandem mass spectrometric analysis of the neutral lipid extract of microbial cells and biofilm communities, respiratory ubiquinone (UQ) (1-methyl-2-isoprenyl-3,4-dimethoxyparabenzoquinone) isoprenologues can be separated isocratically in minutes and assayed with a limit of quantification (LOQ) of 9 p.p.b. (11.1 fmol UQ9 microL(-1)). This corresponds to about 1.29 x 10(7) cells of Pseudomonas putida. Highest sensitivity is achieved using flow-injection analysis with multiple reaction monitoring wherein ammoniated molecular ions of specific isoprenologues pass through quadrupole one, are collisionally dissociated in quadrupole two and identified from the product ion fragment at m/z 197.1 in quadrupole three. This assay has a repeatability of between 6% and 10% over three orders of magnitude (r2 = 0.996). Quinone profiling based on dominant isoprenologue patterns provides taxonomic insights. Detection of prominent UQ isoprenologues indicates presence of microeukaryotes and alpha Proteobacteria with UQ10, obligatory aerobic Gram-negative bacteria with UQ4-14, facultative Gram-negative (and some gamma Proteobacteria growing in microniches with oxygen or to a much lesser extent nitrate as a terminal electron acceptor with UQ8, and other gamma Proteobacteria with UQ9. High sensitivity is essential as the phospholipid fatty acid (PLFA) to UQ molar ratios are 130 or greater. Previous studies have established that recovery of sediment communities with high PLFA/UQ ratios corresponded to areas of aerobic metabolism, an important consideration in bioremediation or nuclide mobilization.