Hegeman Adrian D, Schulte Christopher F, Cui Qiu, Lewis Ian A, Huttlin Edward L, Eghbalnia Hamid, Harms Amy C, Ulrich Eldon L, Markley John L, Sussman Michael R
Department of Biochemistry, University of Wisconsin, 433 Babcock Drive, Madison, Wisconsin 53706, USA.
Anal Chem. 2007 Sep 15;79(18):6912-21. doi: 10.1021/ac070346t. Epub 2007 Aug 21.
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. J. Am. Soc. Mass Spectrom. 2000, 11, 835-40) to limit the formulas consistent with any exact mass measurement by comparing observations of metabolites extracted from Arabidopsis thaliana plants grown with (I) (12)C and (14)N (natural abundance), (II) (12)C and (15)N, (III) (13)C and (14)N, or (IV) (13)C and (15)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.
在生物提取物中数千种代谢物的混合物中确定单个化合物的身份是代谢组学技术面临的一项重大挑战。质谱在很大的丰度和分子量动态范围内具有高灵敏度，但在区分同量异位素化合物的能力方面存在局限。在本文中，我们扩展了早期使用同位素标记来阐明元素组成的研究（Rodgers, R. P.; Blumer, E. N.; Hendrickson, C. L.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 2000, 11, 835 - 40），通过比较从生长于（I）（12）C和（14）N（自然丰度）、（II）（12）C和（15）N、（III）（13）C和（14）N或（IV）（13）C和（15）N的拟南芥植物中提取的代谢物的观测结果，来限制与任何精确质量测量一致的分子式。通过使用两种方法分析来自这四种提取物的精确质量测量数据，在显著扩展的质量范围内确定了独特的元素组成。第一种方法是，通过使用针对四种同位素条件各自计算的值，将代谢物质量与已知存在于活细胞中的11,000种化合物的库进行匹配。第二种方法是，针对在所有四种同位素体系中可能的原子组合的受限子集计算的质量，搜索代谢物质量。在这两种方法中，比较每种标记体系的元素组成列表，以找到在四种体系中的至少三种体系中具有相似HPLC保留特性的共同分子式。这些结果表明，代谢标记可用于在扩展的质量范围内为更高置信度的分子式分配提供额外的约束条件。
