脂质在哺乳动物繁殖中起着许多关键的生理作用,对于卵母细胞能力获得和受精后胚胎发育是必不可少的。分析微小样本中的脂质,如卵母细胞,提出了挑战,但已通过质谱技术如多反应监测(MRM)分析完成。通过简化工作流程和检查分析前条件的影响的双重目标,我们评估了室温下的运输是否会影响牛卵母细胞的脂质分布。为此,使用单相(仅甲醇)或双相液体提取方案(Bligh和Dyer方法)制备样品,并在干冰上或室温下在密封的真空包装内运输,以防止脂质氧化。随后,采用全面的方法,我们筛选了来自卵母细胞脂质提取物中10个不同脂质亚类的316个MRM。主成分分析(PCA)揭示了运输过程中温度的相似脂质分布,而根据脂质提取方法观察到样品之间的明显差异。单变量分析表明,单相甲醇提取导致较高的磷脂相对丰度,除了磷脂酰丝氨酸.相反,Bligh&Dyer提取物有利于检测中性细胞内脂质(三酰甘油,游离脂肪酸,胆固醇酯,和酰基肉碱)。因此,脂质回收率与脂质类别的极性和提取方法直接相关。关于运输温度,磷脂酰乙醇胺,三酰基甘油,当样品在室温下运输时,游离脂肪酸的丰度较低。基于多变量和单变量分析,我们得出的结论是,如果样品经过相同的脂质提取方案,并在室温下在真空密封袋中同批运输,分析牛卵母细胞的脂质提取物是可行的,并且仍然可以获得信息丰富的脂质谱分析结果。
Lipids play numerous pivotal physiological roles in mammalian reproduction, being indispensable for oocyte competence acquisition and post-fertilization embryonic development. Profiling lipids in minute samples, such as oocytes, presents challenges but has been accomplished through mass spectrometry technologies like Multiple Reaction Monitoring (MRM) profiling. With the dual objectives of simplifying workflow and examining the influence of preanalytical conditions, we assessed whether transportation at room temperature affects the lipid profile of bovine oocytes. To this end, samples were prepared using either monophasic (methanol only) or biphasic liquid extraction protocols (Bligh & Dyer method) and transported either on dry ice or at room temperature inside sealed-vacuum packages to prevent lipid oxidation. Subsequently, employing a comprehensive method, we screened a list of 316 MRMs from 10 different lipid subclasses in oocyte lipid extracts. Principal Component Analysis (PCA) revealed similar lipid profiles concerning temperature during transportation, whereas clear differentiation among samples was observed based on the lipid extraction method. Univariate analysis indicated that the one-phase methanol extraction resulted in higher relative abundances of phospholipids, except for phosphatidylserines. Conversely, the Bligh & Dyer extraction favored the detection of neutral intracellular lipids (triacylglycerols, free fatty acids, cholesteryl esters, and acyl-carnitines). Consequently, lipid recovery was directly correlated with the polarity of lipid class and the extraction method. Regarding transportation temperature, phosphatidylethanolamine, triacylglycerol, and free fatty acids exhibited lower abundances when samples were transported at room temperature. Based on multivariate and univariate analyses, we conclude that if samples undergo the same lipid extraction protocol and are transported in the same batch at room temperature inside vacuum-sealed bags, it is feasible to analyze lipid extracts of bovine oocytes and still obtain informative lipid profiling results.