10.1021/ac070346t.s002 Adrian D. Hegeman Adrian D. Hegeman Christopher F. Schulte Christopher F. Schulte Qiu Cui Qiu Cui Ian A. Lewis Ian A. Lewis Edward L. Huttlin Edward L. Huttlin Hamid Eghbalnia Hamid Eghbalnia Amy C. Harms Amy C. Harms Eldon L. Ulrich Eldon L. Ulrich John L. Markley John L. Markley Michael R. Sussman Michael R. Sussman Stable Isotope Assisted Assignment of Elemental Compositions for Metabolomics American Chemical Society 2007 HPLC 13 C mass measurement data method 11 000 compounds 14 N mass range Arabidopsis thaliana plants confidence formula assignments 15 N regime Stable Isotope Assisted Assignment III metabolite masses 12 C IV II 2007-09-15 00:00:00 Dataset https://acs.figshare.com/articles/dataset/Stable_Isotope_Assisted_Assignment_of_Elemental_Compositions_for_Metabolomics/2985406 Assignment of individual compound identities within mixtures of thousands of metabolites in biological extracts is a major challenge for metabolomic technology. Mass spectrometry offers high sensitivity over a large dynamic range of abundances and molecular weights but is limited in its capacity to discriminate isobaric compounds. In this article, we have extended earlier studies using isotopic labeling for elemental composition elucidation (Rodgers, R. P.; Blumer, E. N.; Hendrickson, C. L.; Marshall, A. G. <i>J. Am. Soc. Mass Spectrom.</i> <b>2000</b>, <i>11,</i> 835−40) to limit the formulas consistent with any exact mass measurement by comparing observations of metabolites extracted from <i>Arabidopsis thaliana</i> plants grown with (I) <sup>12</sup>C and <sup>14</sup>N (natural abundance), (II) <sup>12</sup>C and <sup>15</sup>N, (III) <sup>13</sup>C and <sup>14</sup>N, or (IV) <sup>13</sup>C and <sup>15</sup>N. Unique elemental compositions were determined over a dramatically enhanced mass range by analyzing exact mass measurement data from the four extracts using two methods. In the first, metabolite masses were matched with a library of 11 000 compounds known to be present in living cells by using values calculated for each of the four isotopic conditions. In the second method, metabolite masses were searched against masses calculated for a constrained subset of possible atomic combinations in all four isotopic regimes. In both methods, the lists of elemental compositions from each labeling regime were compared to find common formulas with similar retention properties by HPLC in at least three of the four regimes. These results demonstrate that metabolic labeling can be used to provide additional constraints for higher confidence formula assignments over an extended mass range.