Combining Amine Metabolomics and Quantitative Proteomics of Cancer Cells Using Derivatization with Isobaric Tags

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.

定量代谢组学与蛋白质组学技术是探索细胞代谢调控机制的强有力研究手段。然而，将两种技术整合应用时，通常需要两套不同的液相色谱-质谱联用（LC-MS）平台，以分别实现代谢物与肽段的高灵敏检测。针对特定类别代谢物的分析优化策略之一，是通过各类衍生化标记物对其进行衍生处理，以提升离子化效率与色谱分离效能。本研究证实，采用常用于多重肽段定量的串联质量标签（TMT）对胺类代谢物进行衍生化，可借助蛋白质组学研究中常用的标准纳流反相液相色谱-质谱联用平台完成氨基酸分析。本研究表明，该策略可通过双工标记物开展一级质谱（MS1）层面的实验，或借助新型含10重报告离子的同量异位素标签实现二级质谱（MS2）层面的胺类代谢物多重定量分析。此外，本研究通过与多重定量蛋白质组学平行开展的实验，依托相同的LC-MS平台实现了氨基酸的绝对定量测定，以此探究细胞内氨基酸的调控机制。本研究进一步证实，该策略还可用于测定葡萄糖碳源参与的细胞内氨基酸代谢标记情况。