In this study we demonstrated the combination of MALDI-TOF MS and TLC as a fast and powerful tool to investigate the phospholipid (PL) composition of organic extracts of bull spermatozoa. Since phosphatidylcholine (PC) is the dominant PL species, an adequate resolution of MALDI-TOF spectra for sphingomyelin (SM) or phosphatidylethanolamine (PE) was achieved only after previous PL separation by TLC. We found a poor diversity especially for PE and PC, mainly containing ether-linked fatty acids which were 1-palmityl-2-docosahexaenoyl-PL and the corresponding alkenyl-acyl compound (plasmalogen) 1-palmitenyl-2-docosahexaenoyl-PL. For PC, both lipids were quantified after phospholipase A2 digestion to represent 44.2 and 37.2%, respectively, of the total PC. In contrast, the diacyl-PC content of bull spermatozoa was comparatively low (18.6% of total PC). In the presence of trifluoroacetic acid (TFA), which is routinely added to the MALDI-TOF matrix to improve the signal to noise ratio, a high lysophospholipid (LPL) content was detected in the PL extracts of bull spermatozoa, whereas TLC did not reveal significant amounts of LPL. The TFA mediated hydrolysis of the acid-labile alkenyl-acyl PL to the corresponding LPL was shown to cause this discrepancy. This assumption was verified by analysing the PL composition by MALDI-TOF MS before and after (i) digestion of sperm cell lipids with phospholipase A2 and (ii) exposition of spermatozoa to HCl fumes. We conclude that the analysis of samples containing alkenyl-acyl-PL by MALDI-TOF has to be performed with great caution.

中文翻译：

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通过MALDI-TOF质谱分析牛精子的脂质成分-注意事项

在这项研究中，我们证明了MALDI-TOF MS和TLC的组合是研究牛精子有机提取物的磷脂（PL）组成的快速而强大的工具。由于磷脂酰胆碱（PC）是主要的PL物质，因此仅在先前通过TLC分离PL后，才能实现鞘磷脂（SM）或磷脂酰乙醇胺（PE）的MALDI-TOF光谱的足够分辨率。我们发现特别是对于PE和PC而言，多样性很差，主要包含以醚键连接的脂肪酸（1-棕榈基-2-十二碳六烯基-PL）和相应的烯基-酰基化合物（血浆原）1-棕榈烯基-2-十二碳六烯基-PL。对于PC，在磷脂酶A2消化后将两种脂质定量，分别代表总PC的44.2％和37.2％。相反，牛精子的二酰基-PC含量相对较低（占总PC的18.6％）。在通常添加到MALDI-TOF基质中以改善信噪比的三氟乙酸（TFA）存在下，在牛精子PL提取物中检测到高溶血磷脂（LPL）含量，而TLC并未显示大量的LPL。TFA介导的酸不稳定的烯基-酰基PL水解为相应的LPL会导致这种差异。通过（i）用磷脂酶A2消化精子细胞脂质和（ii）将精子暴露于HCl烟气之前和之后，通过MALDI-TOF MS分析PL组成，验证了这一假设。我们得出结论，必须非常谨慎地通过MALDI-TOF分析包含烯基-酰基-PL的样品。在牛精子的PL提取物中检测到高的溶血磷脂（LPL）含量，而TLC并未显示出大量的LPL。TFA介导的酸不稳定的烯基-酰基PL水解为相应的LPL会导致这种差异。通过（i）用磷脂酶A2消化精子细胞脂质和（ii）将精子暴露于HCl烟气之前和之后，通过MALDI-TOF MS分析PL组成，验证了这一假设。我们得出结论，必须非常谨慎地通过MALDI-TOF分析包含烯基-酰基-PL的样品。在牛精子的PL提取物中检测到高的溶血磷脂（LPL）含量，而TLC并未显示出大量的LPL。TFA介导的酸不稳定的烯基-酰基PL水解为相应的LPL会导致这种差异。通过（i）用磷脂酶A2消化精子细胞脂质和（ii）将精子暴露于HCl烟气之前和之后，通过MALDI-TOF MS分析PL组成，验证了这一假设。我们得出结论，必须非常谨慎地通过MALDI-TOF分析包含烯基-酰基-PL的样品。TFA介导的酸不稳定的烯基-酰基PL水解为相应的LPL会导致这种差异。通过（i）用磷脂酶A2消化精子细胞脂质和（ii）将精子暴露于HCl烟气之前和之后，通过MALDI-TOF MS分析PL组成，验证了这一假设。我们得出结论，必须非常谨慎地通过MALDI-TOF分析包含烯基-酰基-PL的样品。TFA介导的酸不稳定的烯基-酰基PL水解为相应的LPL会导致这种差异。通过（i）用磷脂酶A2消化精子细胞脂质和（ii）将精子暴露于HCl烟气之前和之后，通过MALDI-TOF MS分析PL组成，验证了这一假设。我们得出结论，必须非常谨慎地通过MALDI-TOF分析包含烯基-酰基-PL的样品。