Nano-electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) was applied to identify the molecular species of phosphatidylethanolamine of Caenorhabditis elegans, which has a high concentration of phospholipids with a fatty acyl chain having an odd number of carbon atoms. The molecular species of diacyl phosphatidylethanolamine with one fatty acyl chain having an odd number of carbon atoms and one fatty acyl chain having an even number of carbon atoms was identified separately from alkyl-acyl phosphatidylethanolamine with an alkyl chain having an even number of carbon atoms and a fatty acyl chain having an even number of carbon atoms. Furthermore, nano-ESI-FTICRMS was applied to the direct identification of oxidized phosphatidylcholine from soybean. The mass peaks of individual molecular species of oxidative phosphatidylcholine, such as 34:3 diacyl phosphatidylcholine with peroxide (+2O) (m/z 788.544) or 34:2 diacyl phosphatidylcholine with peroxide (+2O) (m/z 790.560), can be separated from the peaks of the molecular species of the non-oxidative phospholipids. This suggests that the mass peaks with a difference of less than 0.1 mass units in their molecular weight can be separated and that their individual exact molecular compositions can be obtained by the FTICRMS analysis. The high resolution and high accuracy of FTICRMS are very effective in the analysis of molecular species of phospholipids and their derivatives.

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SummaryAutoantibodies characteristic for anti‐phospholipid syndrome (APS) and systemic lupus erythematosus (SLE) are anti‐β2‐glycoprotein I (β2GPI) antibodies and anti‐DNA antibodies, respectively, and almost half of APS cases occur in SLE. Anti‐β2GPI antibodies are recognized to play a pivotal role in inducing a prothrombotic state, but the precise mechanism has not been fully elucidated. In a widely accepted view, binding of anti‐β2GPI antibodies to cell surface β2GPI in monocytes and endothelial cells triggers the Toll‐like receptor 4‐myeloid differentiation primary response 88 (TLR)‐4‐MyD88) signaling pathway which leads to activation of p38 mitogen‐activated protein kinase (MAPK), mitogen‐activated protein kinase kinase 1/extracellular signal‐regulated kinases (MEK‐1/ERK) and/or nuclear factor kappa B (NF‐κB) and expression of tissue factor (TF). However, resting cells do not express substantial amounts of TLR‐4. Previously, we generated a mouse monoclonal anti‐β2GPI antibody WB‐6 and showed that it induced a prothrombotic state – including TF expression on circulating monocytes – in normal mice. In the current study, we aimed to clarify the mechanism of interaction between WB‐6 and resting monocytes, and found that WB‐6 exhibits binding activity to DNA and enters living monocytes or a monocytic cell line and, to a lesser extent, vascular endothelial cells. Treatment of the cells with DNase I reduced the internalization, suggesting the involvement of cell surface DNA in this phenomenon. Monocytes harboring internalized WB‐6 expressed TF and tumor necrosis factor (TNF)‐α which, in turn, stimulated endothelial cells to express intercellular adhesion molecule 1 (ICAM‐I) and vascular cell adhesion molecule 1 (VCAM‐I). These results suggest the possibility that a subset of anti‐β2GPI antibodies with dual reactivity to DNA possesses ability to stimulate DNA sensors in the cytoplasm, in addition to the cell surface receptor‐mediated pathways, leading to produce proinflammatory and prothrombotic states.

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