Divito Erin B, Kroniser Kristin M, Cascio Michael
Department of Chemistry and Biochemistry, Duquesne University Pittsburgh, PA, USA.
Front Physiol. 2016 Dec 15;7:608. doi: 10.3389/fphys.2016.00608. eCollection 2016.
Recognition of the contributions of lipids to cellular physiology, both as structural components of the membrane and as modulatory ligands for membrane proteins, has increased in recent years with the development of the biophysical and biochemical tools to examine these effects. Their modulatory roles in ion channels and transporters function have been extensively characterized, with the molecular mechanisms of these activities being the subject of intense scrutiny. The physiological significance of lipids in biochemistry is expanding as numerous fatty acyls are discovered to possess signaling properties. These bioactive lipids are often found in quantities of pmol/g of tissue and are co-extracted with numerous lipophilic molecules, making their detection and identification challenging. Common analytical methodologies involve chromatographic separation and mass spectrometric techniques; however, a single chromatographic step is typically ineffective due to the complexity of the biological samples. It is, therefore, essential to develop approaches that incorporate multiple dimensions of separation. Described in this manuscript are normal phase and reversed phase separation strategies for lipids that include detection of the bioactive primary fatty acid amides and N-acyl glycines via tandem mass spectrometry. Concerted utilization of these approaches are then used to separate and sensitively identify primary fatty acid amides extracted from homogenized tissue, using mouse brains as a test case.
近年来，随着用于研究这些效应的生物物理和生化工具的发展，人们越来越认识到脂质在细胞生理学中的作用，脂质既是细胞膜的结构成分，也是膜蛋白的调节配体。它们在离子通道和转运体功能中的调节作用已得到广泛表征，这些活动的分子机制是深入研究的主题。随着大量脂肪酰基被发现具有信号特性，脂质在生物化学中的生理意义正在不断扩展。这些生物活性脂质通常以每克组织皮摩尔的量存在，并与众多亲脂性分子一起被共提取，这使得它们的检测和鉴定具有挑战性。常见的分析方法包括色谱分离和质谱技术；然而，由于生物样品的复杂性，单一的色谱步骤通常效果不佳。因此，开发包含多个分离维度的方法至关重要。本手稿描述了脂质的正相和反相分离策略，包括通过串联质谱检测生物活性伯脂肪酸酰胺和N-酰基甘氨酸。然后，以小鼠脑为测试案例，协同利用这些方法分离并灵敏鉴定从匀浆组织中提取的伯脂肪酸酰胺。
