研究目的
To develop a large-scale lipid analysis platform for localizing carbon–carbon double bonds (C=C) in unsaturated lipids to distinguish C=C location isomers, which is challenging in conventional lipidomics, and to apply this for biomarker discovery in diseases like breast cancer and type 2 diabetes.
研究成果
The LC-PB-MS/MS platform enables comprehensive structural analysis of unsaturated phospholipids at the C=C location level, identifying over 200 lipids and 55 isomer groups in bovine liver. It demonstrates high precision in measuring isomer ratios with reduced interpersonal variations, making it suitable for biomarker discovery in breast cancer and type 2 diabetes, where isomer compositional changes provide more reliable indicators than subclass-level profiling.
研究不足
The study is limited by the mass resolving power of the ion trap instrument, which may not confidently identify ether lipids co-existing with diacyl lipids as minor components. Sensitivity of LC-PB-MS/MS is lower than LC-MS/MS due to loss in conversion of unsaturated lipids to PB products. The method requires prior fatty acyl composition determination for C=C location assignment, and some minor isomers may not be reported due to low signal-to-noise or overlap.
1：Experimental Design and Method Selection:
The study employs an online-coupled LC-PB-MS/MS platform integrating liquid chromatography with Paternò-Büchi (PB) reaction and tandem mass spectrometry. The PB reaction is used for C=C-specific derivatization, and hydrophilic interaction chromatography (HILIC) is chosen for lipid class separation.
2：Sample Selection and Data Sources:
Samples include bovine liver polar lipid extract, human breast cancer tissue samples (6 cancerous and 6 para-carcinoma sections), and human plasma samples from type 2 diabetes patients and controls (6 each). Lipid extraction is performed using a modified Folch method.
3：List of Experimental Equipment and Materials:
Key instruments include an ExionLC AC system (Sciex), a 4500 QTRAP mass spectrometer (Sciex), a home-built flow microreactor made from FEP tubing with a low-pressure mercury lamp, and a HILIC column (150 mm ×
4：1 mm, silica spheres, 7 μm, Sigma-Aldrich). Reagents include acetone, ACN, ammonium acetate, and lipid standards from Avanti Polar Lipids, Inc. Experimental Procedures and Operational Workflow:
The workflow involves LC-MS/MS runs for lipid subclass and fatty acyl level identification, followed by data-dependent LC-PB-MS/MS for C=C location determination. The PB reaction is conducted post-LC separation using UV irradiation in a flow microreactor. Data collection modes include precursor ion scan (PIS), neutral loss scan (NLS), and enhanced product ion (EPI).
5：Data Analysis Methods:
Data analysis is performed using Analyst software and a home-developed program, Lipid Omega Analyzer (LOA), for structural identification at subclass, acyl chain, and C=C location levels. Statistical analysis includes student's t-test and hierarchical clustering.
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