Abstract
Metabolites that incorporate elements other than carbon, nitrogen, hydrogen and oxygen can be selectively detected by inductively coupled mass spectrometry (ICPMS). When used in parallel with chromatographic separations and conventional electrospray ionization mass spectrometry (ESIMS), ICPMS allows the analyst to quickly find, characterize and identify target metabolites that carry nutrient elements (P, S, trace metals; “nutrient metabolites”), which are of particular interest to investigations of microbial biogeochemical cycles. This approach has been applied to the study of siderophores and other trace metal organic ligands in the ocean. The original method used mass search algorithms that relied on the ratio of stable isotopologues of iron, copper and nickel to assign mass spectra collected by ESIMS to metabolites carrying these elements detected by ICPMS. However, while isotopologue-based mass assignment algorithms were highly successful in characterizing metabolites that incorporate some trace metals, they do not realize the whole potential of the ICPMS/ESIMS approach as they cannot be used to assign the molecular ions of metabolites with monoisotopic elements or elements for which the ratio of stable isotopes is not known. Here we report a revised ICPMS/ESIMS method that incorporates a number of changes to the configuration of instrument hardware that improves sensitivity of the method by a factor of 4–5, and allows for more accurate quantitation of metabolites. We also describe a new suite of mass search algorithms that can find and characterize metabolites that carry monoisotopic elements. We used the new method to identify siderophores in a laboratory culture of Vibrio cyclitrophicus and a seawater sample collected in the North Pacific Ocean, and to assign molecular ions to monoisotopic cobalt and iodine nutrient metabolites in extracts of a laboratory culture of the marine cyanobacterium Prochorococcus MIT9215.
Highlights
Advances in the chromatographic separation and mass spectral analyses of metabolites have opened new avenues for understanding microbial dynamics and organic matter cycling in the ocean (Petras et al, 2017; Patriarca et al, 2018)
The comparative analysis of metabolites expressed in particulate or dissolved organic matter sampled under different conditions, is typically approached in one of two ways: through targeted metabolomics in which metabolites of interest are measured through an experiment or suite of samples, and untargeted metabolomics in which the response of unidentified metabolites are correlated against experimental or environmental variables
inductively coupled plasma mass spectrometry (ICPMS) and ESIMS data can be collected in parallel by splitting the outflow of the liquid chromatography (LC) system between the two spectrometers, in practice we found it easier to perform two separate analyses of a sample and align the data using an internal standard
Summary
Advances in the chromatographic separation and mass spectral analyses of metabolites have opened new avenues for understanding microbial dynamics and organic matter cycling in the ocean (Petras et al, 2017; Patriarca et al, 2018). The comparative analysis of metabolites expressed in particulate or dissolved organic matter sampled under different conditions, is typically approached in one of two ways: through targeted metabolomics in which metabolites of interest are measured through an experiment or suite of samples, and untargeted metabolomics in which the response of unidentified metabolites are correlated against experimental or environmental variables (reviewed by Soule et al, 2015; Cajka and Fiehn, 2016). Untargeted approaches survey a much broader suite of metabolites, but in doing so a detailed knowledge of metabolite identity and quantity is sacrificed. These two approaches bookend other avenues for investigating marine metabolomics that share features with both targeted and untargeted analyses. The approach can be adapted and applied to metabolites that share other chemical properties such as UV/Vis light absorption or fluorescence
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