Department of Chemistry and Chemical Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4M1.
Anal Chem. 2023 Dec 5;95(48):17513-17524. doi: 10.1021/acs.analchem.3c02605. Epub 2023 Nov 22.
Orthogonal separation techniques coupled to high-resolution mass spectrometry are required for characterizing the human lipidome, given its inherent chemical and structural complexity. However, electrophoretic separations remain largely unrecognized in contemporary lipidomics research compared to established chromatographic and ion mobility methods. Herein, we introduce a novel derivatization protocol based on 3-methyl-1--tolyltriazene (MTT) as a safer alternative to diazomethane for quantitative phospholipid (PL) methylation (∼90%), which enables their rapid analysis by multisegment injection-nonaqueous capillary electrophoresis-mass spectrometry (MSI-NACE-MS). Isobaric interferences and ion suppression effects were minimized by performing an initial reaction using 9-fluorenylmethyoxycarbonyl chloride prior to MTT and a subsequent back extraction in hexane. This charge-switch derivatization strategy expands lipidome coverage when using MSI-NACE-MS under positive ion mode with improved resolution, greater sensitivity, and higher throughput (∼3.5 min/sample), notably for zwitterionic PLs that are analyzed as their cationic phosphate methyl esters. Our method was validated by analyzing methyl--butyl ether extracts of reference human plasma, which enabled a direct comparison of 48 phosphatidylcholine and 27 sphingomyelin species previously reported in an interlaboratory lipidomics harmonization study. The potential for plasma PL quantification by MSI-NACE-MS via a serial dilution of NIST SRM-1950 was also demonstrated based on estimation of relative response factors using their reported consensus concentrations. Moreover, lipid identification was supported by modeling predictable changes in the electrophoretic mobility for cationic PLs in conjunction with MS/MS. Overall, this work offers a practical derivatization protocol to expand lipidome coverage in CE-MS beyond the analysis of hydrophilic/polar metabolites under aqueous buffer conditions.
鉴于其内在的化学和结构复杂性，需要使用正交分离技术与高分辨率质谱相结合来对人类脂质组进行特征分析。然而，与已建立的色谱和离子淌度方法相比，电泳分离在当代脂质组学研究中仍然很大程度上未得到认可。在此，我们引入了一种基于 3-甲基-1-（对甲苯基）三氮烯（MTT）的新型衍生化方案，作为定量磷脂（PL）甲基化（约 90%）的更安全替代物，可通过多段注射非水毛细管电泳-质谱联用（MSI-NACE-MS）快速分析。通过在 MTT 之前使用 9-芴甲氧羰基氯进行初始反应，然后在正己烷中进行后续反萃取，可以最大程度地减少等排干扰和离子抑制效应。在正离子模式下使用 MSI-NACE-MS 时，这种电荷转换衍生化策略可扩展脂质组覆盖范围，提高分辨率、灵敏度和通量（约 3.5 分钟/样品），对于分析为阳离子磷酸甲酯的两性离子 PL 尤其如此。通过分析参考人血浆的甲基-丁醚提取物对我们的方法进行了验证，该方法可直接比较先前在实验室间脂质组学协调研究中报道的 48 种磷脂酰胆碱和 27 种鞘磷脂。还通过使用其报告的共识浓度来估计相对响应因子，基于 NIST SRM-1950 的系列稀释来演示通过 MSI-NACE-MS 对血浆 PL 进行定量的可能性。此外，通过结合 MS/MS 对阳离子 PL 的电泳迁移率的可预测变化进行建模，支持脂质的鉴定。总体而言，这项工作提供了一种实用的衍生化方案，可在 CE-MS 中扩展脂质组覆盖范围，超越在水缓冲条件下分析亲水性/极性代谢物。
