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. 2020 Apr 7;9:e53582. doi: 10.7554/eLife.53582

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© 2020, Miyamoto et al

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Figure 4. — (A, B) Product ion scanning of the OAHFA (O-C18:1)-ω-OH C22:0 FA was performed by LC-MS/MS by selecting the [M–H]^– ion, with m/z 619.5, as a precursor ion. The MS spectrum (A) and the predicted cleavage positions (B) are shown. A synthesis scheme for OAHFA [(O-C18:1)-ω-OH C22:0 FA] is provided in Figure 4—figure supplement 1. (C–F) Lipids were extracted from the meibomian glands of 12-month-old control (Tg-Cyp4f39^+/+ [n = 2] and Tg-Cyp4f39^+/– [n = 1]) and Tg-Cyp4f39^–/– (n = 3) mice. After derivatization with AMPP, OAHFAs containing C16:0 (F), C16:1 (C, F), C18:0 (F), C18:1 (D, F), and C18:2 (E, F) FA were analyzed by LC-MS/MS. The peak areas for OAHFA species that have different FA chain lengths and degrees of unsaturation (saturated or monounsaturated; panels [C–E]), and their total amounts (F) are shown. Values presented are means ± SD (*, p<0.05; **, p<0.01; Student’s t-test). The simplified structure for each OAHFA is shown below the graph. (G) Lipids extracted from the meibomian glands of 6–12-month-old Tg-Cyp4f39^+/+ (n = 3) and Tg-Cyp4f39^–/– mice (n = 3) were subjected to alkaline treatment and AMPP derivatization, and C16:0, C16:1, C18:0, C18:1, and C18:2 FAs were quantified using LC-MS/MS. Values are presented as means ± SD. nd, not detected; Tg –/–, Tg-Cyp4f39^–/–.

Figure 4—figure supplement 1. — (A, B) Product ion scanning of the OAHFA (O-C18:1)-ω-OH C22:0 FA was performed by LC-MS/MS by selecting the [M–H]^– ion, with m/z 619.5, as a precursor ion. The MS spectrum (A) and the predicted cleavage positions (B) are shown. A synthesis scheme for OAHFA [(O-C18:1)-ω-OH C22:0 FA] is provided in Figure 4—figure supplement 1. (C–F) Lipids were extracted from the meibomian glands of 12-month-old control (Tg-Cyp4f39^+/+ [n = 2] and Tg-Cyp4f39^+/– [n = 1]) and Tg-Cyp4f39^–/– (n = 3) mice. After derivatization with AMPP, OAHFAs containing C16:0 (F), C16:1 (C, F), C18:0 (F), C18:1 (D, F), and C18:2 (E, F) FA were analyzed by LC-MS/MS. The peak areas for OAHFA species that have different FA chain lengths and degrees of unsaturation (saturated or monounsaturated; panels [C–E]), and their total amounts (F) are shown. Values presented are means ± SD (*, p<0.05; **, p<0.01; Student’s t-test). The simplified structure for each OAHFA is shown below the graph. (G) Lipids extracted from the meibomian glands of 6–12-month-old Tg-Cyp4f39^+/+ (n = 3) and Tg-Cyp4f39^–/– mice (n = 3) were subjected to alkaline treatment and AMPP derivatization, and C16:0, C16:1, C18:0, C18:1, and C18:2 FAs were quantified using LC-MS/MS. Values are presented as means ± SD. nd, not detected; Tg –/–, Tg-Cyp4f39^–/–.