Ulmer Candice Z, Yost Richard A, Chen Jing, Mathews Clayton E, Garrett Timothy J
Department of Chemistry, University of Florida, Gainesville, FL, USA.
Department of Chemistry, University of Florida, Gainesville, FL, USA ; Department of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, FL, USA.
J Proteomics Bioinform. 2015 Jun;8(6):126-132. doi: 10.4172/jpb.1000360.
Metabolomics is the comprehensive study of metabolism as it pertains to an organism or biological system. Lipidomics, a subset discipline of metabolomics, encompasses the study of cellular lipid functions: including pathways, networks, and interactions. The abundance of metabolites and lipids, along with their contribution to health and disease, makes metabolomics a valuable tool for biomarker research. Disease biomarker identification requires a reproducible, sensitive, and accurate analytical platform. Although transcriptomic and proteomic areas have well-established protocols for sample preparation and data processing, the metabolomics field is still developing comparable standardized conventions. Furthermore, of the few comparative LC-MS metabolomic studies that have been applied to mammalian cell cultures, most are targeted to adherent cell lines. The purpose of this work was to optimize a sample preparation workflow for the cellular metabolomic analysis of suspension-cultured mammalian cells using commercially available Jurkat T lymphocytes as a model system. The current investigation evaluated commonly used sample preparation techniques for reproducibility, accuracy, and applicability to untargeted biomarker discovery. Results show ammoniated cell rinsing solutions to be an effective means to remove extracellular components present in the media without causing ion suppression or affecting the integrity of the cellular membrane. Additionally, a novel workflow was designed to allow for the combined analysis of metabolites and lipids from mammalian suspension cells from a single cell pellet. The Folch lipid extraction protocol was coupled to an 80% MeOH metabolite isolation to ensure high extraction efficiency for phospholipids and triacylglycerides. While the workflow was tailored to cells in suspension, it could also be applied to adherent cell lines.
代谢组学是对与生物体或生物系统相关的代谢过程进行的全面研究。脂质组学作为代谢组学的一个子学科，涵盖了对细胞脂质功能的研究：包括代谢途径、网络和相互作用。代谢物和脂质的丰度，以及它们对健康和疾病的影响，使代谢组学成为生物标志物研究的宝贵工具。疾病生物标志物的鉴定需要一个可重复、灵敏且准确的分析平台。尽管转录组学和蛋白质组学领域已经有完善的样品制备和数据处理方案，但代谢组学领域仍在开发类似的标准化方法。此外，在已应用于哺乳动物细胞培养的少数比较性液相色谱 - 质谱代谢组学研究中，大多数针对的是贴壁细胞系。这项工作的目的是优化一种样品制备流程，以使用市售的Jurkat T淋巴细胞作为模型系统，对悬浮培养的哺乳动物细胞进行细胞代谢组学分析。当前的研究评估了常用的样品制备技术在可重复性、准确性以及对非靶向生物标志物发现的适用性。结果表明，氨化细胞冲洗溶液是去除培养基中细胞外成分的有效方法，不会导致离子抑制或影响细胞膜的完整性。此外，还设计了一种新颖的流程，以便对来自单个细胞沉淀的哺乳动物悬浮细胞中的代谢物和脂质进行联合分析。Folch脂质提取方案与80%甲醇代谢物分离方法相结合，以确保对磷脂和甘油三酯的高提取效率。虽然该流程是针对悬浮细胞定制的，但也可应用于贴壁细胞系。
