Murphy J Patrick, Everley Robert A, Coloff Jonathan L, Gygi Steven P
Department of Cell Biology, Harvard Medical School , Boston, Massachusetts 02115, United States.
Anal Chem. 2014 Apr 1;86(7):3585-93. doi: 10.1021/ac500153a. Epub 2014 Mar 20.
Quantitative metabolomics and proteomics technologies are powerful approaches to explore cellular metabolic regulation. Unfortunately, combining the two technologies typically requires different LC-MS setups for sensitive measurement of metabolites and peptides. One approach to enhance the analysis of certain classes of metabolites is by derivatization with various types of tags to increase ionization and chromatographic efficiency. We demonstrate here that derivatization of amine metabolites with tandem mass tags (TMT), typically used in multiplexed peptide quantitation, facilitates amino acid analysis by standard nanoflow reversed-phase LC-MS setups used for proteomics. We demonstrate that this approach offers the potential to perform experiments at the MS1-level using duplex tags or at the MS2-level using novel 10-plex reporter ion-containing isobaric tags for multiplexed amine metabolite analysis. We also demonstrate absolute quantitative measurements of amino acids conducted in parallel with multiplexed quantitative proteomics, using similar LC-MS setups to explore cellular amino acid regulation. We further show that the approach can also be used to determine intracellular metabolic labeling of amino acids from glucose carbons.
定量代谢组学和蛋白质组学技术是探索细胞代谢调控的有力方法。不幸的是，结合这两种技术通常需要不同的液相色谱-质谱（LC-MS）设置来灵敏地测量代谢物和肽段。增强某些类代谢物分析的一种方法是用各种类型的标签进行衍生化，以提高离子化和色谱效率。我们在此证明，通常用于多重肽定量的串联质量标签（TMT）对胺类代谢物进行衍生化，有助于通过用于蛋白质组学的标准纳流反相LC-MS设置进行氨基酸分析。我们证明，这种方法有可能使用双链标签在MS1水平或使用新型含10重报告离子的等压标签在MS2水平进行实验，以进行多重胺类代谢物分析。我们还展示了与多重定量蛋白质组学并行进行的氨基酸绝对定量测量，使用类似的LC-MS设置来探索细胞氨基酸调控。我们进一步表明，该方法还可用于确定来自葡萄糖碳的氨基酸的细胞内代谢标记。
