Green C R, Kolar M J, McGregor G H, Nelson A T, Wallace M, Metallo C M
Molecular and Cellular Biology Laboratory, The Salk Institute for Biological Studies, 10010N. Torrey Pines Rd., La Jolla, 92037, CA, USA.
Department of Dermatology, University of California, San Diego, La Jolla, CA 92037, USA.
bioRxiv. 2024 May 28:2024.05.28.596332. doi: 10.1101/2024.05.28.596332.
Compound lipids comprise a diverse group of metabolites present in living systems, and metabolic- and environmentally-driven structural distinctions across this family is increasingly linked to biological function. However, methods for deconvoluting these often isobaric lipid species are lacking or require specialized instrumentation. Notably, acyl-chain diversity within cells may be influenced by nutritional states, metabolic dysregulation, or genetic alterations. Therefore, a reliable, validated method of quantifying structurally similar even-, odd-, and branched-chain acyl groups within intact compound lipids will be invaluable for gaining molecular insights into their biological functions. Here we demonstrate the chromatographic resolution of isobaric lipids containing distinct combinations of straight-chain and branched-chain acyl groups via ultra-high-pressure liquid chromatography (UHPLC)-mass spectrometry (MS) using a C30 liquid chromatography column. Using metabolically-engineered adipocytes lacking branched-keto acid dehydrogenase A (Bckdha), we validate this approach through a combination of fatty acid supplementation and metabolic tracing using monomethyl branched-chain fatty acids and valine. We observe resolution of numerous isobaric triacylglycerols and other compound lipids, demonstrating the resolving utility of this method. This approach strengthens our ability to quantify and characterize the inherent diversity of acyl chains across the lipidome.
复合脂质是存在于生物系统中的一类多样的代谢物，该家族中由代谢和环境驱动的结构差异与生物学功能的联系日益紧密。然而，用于解卷积这些通常为等压脂质种类的方法尚缺，或需要专门的仪器设备。值得注意的是，细胞内的酰基链多样性可能受营养状态、代谢失调或基因改变的影响。因此，一种可靠的、经过验证的方法，用于定量完整复合脂质中结构相似的偶数、奇数和支链酰基，对于深入了解其生物学功能的分子机制将具有重要价值。在此，我们展示了通过使用C30液相色谱柱的超高压液相色谱（UHPLC）-质谱（MS），对含有直链和支链酰基不同组合的等压脂质进行色谱分离。利用缺乏支链酮酸脱氢酶A（Bckdha）的代谢工程化脂肪细胞，我们通过补充脂肪酸和使用单甲基支链脂肪酸及缬氨酸进行代谢示踪相结合的方式验证了该方法。我们观察到众多等压三酰甘油和其他复合脂质的分离效果，证明了该方法的分离效用。这种方法增强了我们定量和表征脂质组中酰基链固有多样性的能力。
