Riusech Olga, Hao Ling, Li Lingjun
Department of Chemistry, University of Wisconsin, Madison, WI, USA.
Department of Chemistry & Biochemistry, University of Maryland, 8051 Regents Dr., College Park, MD, 20742, USA.
Anal Bioanal Chem. 2025 May;417(13):2797-2805. doi: 10.1007/s00216-025-05748-9. Epub 2025 Jan 24.
Quantitative measurement of metabolites is essential to understand biological and disease processes. Absolute quantification by spiking heavy isotope-labeled internal standards and analyzing on mass spectrometry (MS) platform is a key method to determine the concentration of metabolites in biological samples. However, MS-based absolute quantification is often challenged by the commercial availability and high costs of isotope-labeled internal standards. Here, we establish an absolute quantification method for amine metabolites utilizing isotopic N,N-dimethyl leucine (iDiLeu) tagging on the LC-MS/MS platform. Absolute quantification of metabolites with excellent accuracy and precision can be achieved with five-plex iDiLeu labeling without the need of isotope-labeled internal standards. We demonstrated that iDiLeu labeling improved the separation and detection limits of polar metabolites. Particularly, detection limits for glycine, GABA, and serotonin have been improved by more than 20 folds, and valine by more than 2000 folds. With iDiLeu tagging, 87 amine-containing metabolites were identified and quantified in human cerebrospinal fluid (CSF) samples, revealing potential metabolic changes in Alzheimer's disease patients.
代谢物的定量测量对于理解生物和疾病过程至关重要。通过添加重同位素标记的内标并在质谱（MS）平台上进行分析来进行绝对定量，是确定生物样品中代谢物浓度的关键方法。然而，基于MS的绝对定量常常受到同位素标记内标的商业可得性和高成本的挑战。在此，我们在LC-MS/MS平台上利用同位素N，N-二甲基亮氨酸（iDiLeu）标记建立了一种胺类代谢物的绝对定量方法。通过五重iDiLeu标记可实现对代谢物的高精度和高准确度的绝对定量，而无需同位素标记的内标。我们证明iDiLeu标记提高了极性代谢物的分离度和检测限。特别是，甘氨酸、γ-氨基丁酸和血清素的检测限提高了20多倍，缬氨酸的检测限提高了2000多倍。通过iDiLeu标记，在人脑脊液（CSF）样本中鉴定并定量了87种含胺代谢物，揭示了阿尔茨海默病患者潜在的代谢变化。
