Utilizing the total phosphorus content of lipid extracts as a normalization strategy in quantitative lipidomics studies

IF 5.7 2区化学 Q1 CHEMISTRY, ANALYTICAL

Analytica Chimica Acta Pub Date : 2025-05-20 DOI:10.1016/j.aca.2025.344228

Johannes Scholz , Anna Gremme , Julia Hillebrand , Jasper Baldauf , Lennart J. Bendheuer , Jonas Berreiter , Anne C. Sehnal , Julia Bornhorst , Heiko Hayen

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Abstract

Background

Normalization is a crucial element of quantitative lipidomics studies, for which there is still no uniform recommended strategy. There are various approaches for normalizing samples, including referencing to the total volume or weight, to the DNA content or the protein content. In addition, samples can be normalized to the phosphorus content, but either not all phospholipid classes are included or a less sensitive colorimetric assay is used. It is therefore necessary to develop new ways of normalization that are both specific and sensitive to compensate for the biological sample variance and the variance from pre-analytical and analytical steps.

Results

We present a tailored normalization method for lipid quantification which is based on the total phosphorus content (TPC) of lipid extracts. The TPC reflects the amount of phospholipids present in the sample, because polar phosphorus-containing compounds like DNA and phosphorylated sugars are separated due to the extraction procedure. In order to test the normalization strategy, the (phospho)-lipids in the model organism C. elegans were quantified using SFC-TIMS-MS/MS. Subsequently, a digestion method was developed to degrade the lipids present in the organic extract. This was used to determine the TPC via ICP-OES and ICP-MS/MS analysis. In particular, very low detection limits (LOQ = 0.3 μg L⁻¹) can be achieved with the ICP-MS/MS method. The elemental and molecular information were then combined, and the samples were normalized to TPC. The normalization showed a significant improvement in the standard deviations compared to the initial concentrations as well as an improvement compared to the normalization based on the total protein content, which is very frequently used.

Significance

Our method shows a sensitive way to determine the TPC in total lipid extracts of various samples, exemplified for human plasma and nematode samples. The normalization minimizes the standard deviations in biological samples even in comparison with other normalization approaches, i.e. protein content, and can contribute to increasing the conclusiveness of quantitative lipidomics studies.

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利用脂质提取物的总磷含量作为定量脂质组学研究的规范化策略

归一化是定量脂组学研究的关键因素，目前尚无统一的推荐策略。有各种归一化样品的方法，包括参照总体积或重量、DNA含量或蛋白质含量。此外，样品可以归一化到磷含量，但要么不包括所有磷脂类，要么使用敏感度较低的比色法测定。因此，有必要开发新的归一化方法，既具体又敏感，以补偿生物样本方差和分析前和分析步骤的方差。结果提出了一种基于总磷含量（TPC）的脂质定量归一化方法。TPC反映了样品中存在的磷脂的数量，因为极性含磷化合物如DNA和磷酸化糖在提取过程中被分离。为了验证归一化策略，采用SFC-TIMS-MS/MS对模式生物秀丽隐杆线虫（C. elegans）的（磷酸）脂质进行定量。随后，开发了一种消化方法来降解有机提取物中存在的脂质。采用ICP-OES和ICP-MS/MS分析测定TPC。其中ICP-MS/MS方法的检出限极低（LOQ=0.2 μg L-1）。然后结合元素和分子信息，将样品归一化为TPC。与初始浓度相比，归一化在标准偏差方面有显著改善，与基于总蛋白质含量的归一化相比也有改善，这是非常常用的。意义本方法可灵敏地测定各种样品的总脂质提取物中的TPC，以人血浆和线虫样品为例。即使与其他归一化方法（即蛋白质含量）相比，这种归一化方法也将生物样品中的标准偏差降至最低，并有助于提高定量脂质组学研究的结论性。

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来源期刊

Analytica Chimica Acta 化学-分析化学

CiteScore

10.40

自引率

6.50%

发文量

1081

审稿时长

38 days

期刊介绍： Analytica Chimica Acta has an open access mirror journal Analytica Chimica Acta: X, sharing the same aims and scope, editorial team, submission system and rigorous peer review. Analytica Chimica Acta provides a forum for the rapid publication of original research, and critical, comprehensive reviews dealing with all aspects of fundamental and applied modern analytical chemistry. The journal welcomes the submission of research papers which report studies concerning the development of new and significant analytical methodologies. In determining the suitability of submitted articles for publication, particular scrutiny will be placed on the degree of novelty and impact of the research and the extent to which it adds to the existing body of knowledge in analytical chemistry.