A Procedure for Solid-Phase Extractions Using Metal-Oxide-Coated Silica Column in Lipidomics

Lipid
enrichment is indispensable for enhancing the coverage
of
targeted molecules in mass spectrometry (MS)-based lipidomics studies.
In this study, we developed a simple stepwise fractionation method
using a titanium- and zirconium-dioxide-coated solid-phase extraction
(SPE) silica column that separates neutral lipids, phospholipids,
and other lipids, including fatty acids (FAs) and glycolipids. Chloroform
was used to dissolve the lipids, and neutral lipids, including steryl
esters,
diacylglycerols, and triacylglycerols, were collected in the loading
fraction. Second, methanol with formic acid (99:1, v/v) was used to
retrieve FAs, ceramides, and glycolipids, including glycosylated ceramides
and glycosylated diacylglycerols, by competing for affinity with the
Lewis acid sites on the metal oxide surface. Finally, phospholipids
strongly retained via chemoaffinity interactions were eluted using
a solution containing 5% ammonia and high water content (45:50 v/v,
2-propanol:water), which canceled the electrostatic and chelating
interactions with the SPE column. High average reproducibility of
<10% and coverage of ∼100% compared to those of the non-SPE
samples were demonstrated by untargeted lipidomics of human plasma
and mouse brain, testis, and feces. The advantage of our procedure
was showcased by characterizing minor lipid subclasses, including
dihexosylceramides containing very long-chain polyunsaturated FA in
the testis, monogalactosyl and digalactosyl monoacylglycerols in feces,
and acetylated and glycolylated derivatives of gangliosides in the
brain that were not detected using conventional solvent extraction
methods. Likewise, the value of our method in biology is maximized
during glycolipidome profiling in the absence of neutral lipids and
phospholipids that cover more than 80% of the chromatographic peaks.

脂质富集对于提升基于质谱（MS）的脂质组学研究中靶向分子的覆盖度至关重要。本研究开发了一种简便的分步分级分离方法，采用涂覆二氧化钛与二氧化锆的固相萃取（SPE）硅胶柱，可分离中性脂质、磷脂以及包括脂肪酸（FAs）和糖脂在内的其他脂质类群。以三氯甲烷溶解脂质样品，上样组分中可收集到甾醇酯、二酰甘油及三酰甘油等中性脂质。第二步，采用体积比为99:1的甲酸甲醇溶液，通过与金属氧化物表面的路易斯酸位点竞争结合作用，洗脱收集脂肪酸、神经酰胺以及包括糖基化神经酰胺和糖基化二酰甘油在内的糖脂类物质。最后，通过化学亲和作用紧密结合的磷脂，可使用含5%氨水的高水相溶液（体积比45:50的异丙醇-水溶液）洗脱，该溶液可破坏其与SPE柱的静电及螯合相互作用。通过对人血浆以及小鼠脑、睾丸和粪便样品进行非靶向脂质组学分析，本方法相较于非SPE处理样品，实现了平均相对标准偏差低于10%的高重现性，以及约100%的脂质覆盖度。本方法的优势通过对微量脂质亚类的表征得以体现：例如在睾丸样品中鉴定出含超长链多不饱和脂肪酸的二己糖基神经酰胺，在粪便样品中检测到单半乳糖基单酰甘油与二半乳糖基单酰甘油，而在脑组织中鉴定出常规溶剂萃取法无法检出的神经节苷脂乙酰化及糖基化衍生物。此外，在中性脂质与磷脂占据色谱峰总量80%以上的样品中开展糖脂组分析时，本方法可去除绝大多数中性脂质与磷脂，从而最大化其在生物学研究中的应用价值。