Synthesis and Use of Stable-Isotope-Labeled Internal Standards for Quantification of Phosphorylated Metabolites by LC鈥揗S/MS

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• 作者：St茅phanie Arrivault ; Manuela Guenther ; Stephen C. Fry ; Maximilian M. F. F. Fuenfgeld ; Daniel Veyel ; Tabea Mettler-Altmann ; Mark Stitt ; John E. Lunn
• 刊名：Analytical Chemistry
• 出版年：2015
• 出版时间：July 7, 2015
• 年：2015
• 卷：87
• 期：13
• 页码：6896-6904
• 全文大小：465K
• ISSN：1520-6882

文摘

Liquid chromatography coupled to tandem mass spectrometry (LC鈥揗S/MS) is a highly specific and sensitive technique for measuring metabolites. However, coeluting components in tissue extracts can interfere with ionization at the interface of the LC and MS/MS phases, causing under- or overestimation of metabolite concentrations. Spiking of samples with known amounts of stable-isotope-labeled internal standards (SIL-IS) allows measurements of the corresponding metabolites to be corrected for such matrix effects. We describe criteria for selection of suitable SIL-IS and report the enzymatic synthesis and purification of nine SIL-IS for hexose-, pentose-, and triose-phosphates, UDP-glucose, and adenosine monophosphate (AMP). Along with commercially available SIL-IS for seven other metabolites, these were validated by LC鈥揗S/MS analyses of extracts from leaves, nonphotosynthetic plant tissues, mouse liver, and cells of Chlamydomonas reinhardtii, Escherichia coli and baker鈥檚 yeast (Saccharomyces cerevisiae). With only a few exceptions, spiking with SIL-IS significantly improved the reproducibility of LC鈥揗S/MS-based metabolite measurements across a wide range of extract dilutions, indicating effective correction for matrix effects by this approach. With use of SIL-IS to correct for matrix effects, LC鈥揗S/MS offers unprecedented scope for reliable determination of photosynthetic and respiratory intermediates in a diverse range of organisms.