![Separation of HexCer diastereomers. (A) MRM chromatograms of the synthesized four diastereomers of HexCer (d18:1/16:0) and (d18:1/24:1) were obtained using SFC/MRM. SFC separated the four diastereomers according to their RTs even when mixed (bottom). MRM transitions were 700.57 > 264.27 for HexCer (d18:1/16:0) and 810.68 > 264.27 for HexCer (d18:1/24:1), (precursor ions were selected as [M+H]+). The colored shadows indicate the peaks coincident with the RT of synthesized α-GalCer (red), α-GlcCer (blue), β-GalCer (green), and β-GlcCer (yellow), respectively. (B) CD1d−/− or CD1d-expressing DN32.D3 cells were stimulated with the C:M = 19:1 SFC-FRC separated fractions from serum lipids for 16 h and analyzed for CD69 expression. The C:M = 19:1 fraction (10 μg) of serum lipids was separated using SFC, and 1/25th of the product was used for 9.5–13.5 min subfractions and 1/75th of the product was used for the other four sub-fractions (0–9.5 [#1], 13.5–24 [#2], 24–34.5 [#3] and 34.5–45 min [#4]) (Fig. S2 A). (C) HRMS chromatogram of HexCer (d18:0/16:0) in serum. The colored shadows indicate the peaks coincident with the estimated RT of α-GalCer (d18:0/16:0) (red) and β-GlcCer (d18:0/16:0) (yellow), respectively. (D) HRMS-EIC chromatograms of the synthesized four diastereomers of HexCer (d18:0/16:0) obtained using SFC/HRMS. SFC separated the four diastereomers according to their RTs. The red shadow indicates the peak coincident with the RT of synthesized α-GalCer (d18:0/16:0). (E) HRMS/MS spectra of candidates for α-GalCer (d18:0/16:0) in serum. From top to bottom, collision energy settings are −10 eV (+), −20 eV (++), −30 eV (+++), and −40 eV (++++). The HRMS/MS of precursor-product ion pair: m/z 702.5878 to 540.5350, 522.5245, 284.2948, and 266.2842, respectively. (F) HRMS chromatograms of HexCer (d18:0/16:0) and the HRMS/MS spectra of candidates for α-GalCer (d18:0/16:0) in serum spiked with synthesized α-GalCer (d18:0/16:0). From left to right, collision energy settings are −10 eV (+), −20 eV (++), −30 eV (+++), and −40 eV (++++). The structures of product ions are shown. The red shadow indicates the peak coincident with the RT of synthesized α-GalCer (d18:0/16:0). The mass error tolerance of the precursor ion (m/z 702.5878 as [M+H]+) was <7 ppm (E and F). The HRMS/MS of precursor-product ion pair: m/z 702.5878 to 540.5350, 522.5245, 284.2948, and 266.2842, respectively (E and F). Data are presented as mean ± SD (B) and are representative of three independent experiments (A, C, and D–F).](https://cdn.rupress.org/rup/content_public/journal/jem/222/2/10.1084_jem.20240728/1/jem_20240728_fig2.png?Expires=1767647730&Signature=YoUKAczR4iNdXDu2PuqWZnMe2bjb7ACboQ~7kPujR1SBSuzPq~I0zE8oOIoEbKGn-r6eTb-M4pq03zHAMB67KUbZW~rCPFB00ip3OmaR1mgGFwIN0jgVcieWV6ql3-f0ZD0XAaB57tG34b0Mcg10dpKnJKKYrFBbnNTOdyyVYrDPPQ4BXCBf4QfIx-rN4L2YDDtTWewMniv0uDnbR20I7-FPb1i1bPuhY0j8fKhERiQPZRLHCbVkGJ0QDjtufYnIR5-EPh-XsBuqabosEs3k6yECDschN3bWVofc4QNFVC~LbgGFN3Q1Ac~OR-m5HMDBLgQiatZRdR1~VPvJLPFIhg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Separation of HexCer diastereomers. (A) MRM chromatograms of the synthesized four diastereomers of HexCer (d18:1/16:0) and (d18:1/24:1) were obtained using SFC/MRM. SFC separated the four diastereomers according to their RTs even when mixed (bottom). MRM transitions were 700.57 > 264.27 for HexCer (d18:1/16:0) and 810.68 > 264.27 for HexCer (d18:1/24:1), (precursor ions were selected as [M+H]+). The colored shadows indicate the peaks coincident with the RT of synthesized α-GalCer (red), α-GlcCer (blue), β-GalCer (green), and β-GlcCer (yellow), respectively. (B) CD1d−/− or CD1d-expressing DN32.D3 cells were stimulated with the C:M = 19:1 SFC-FRC separated fractions from serum lipids for 16 h and analyzed for CD69 expression. The C:M = 19:1 fraction (10 μg) of serum lipids was separated using SFC, and 1/25th of the product was used for 9.5–13.5 min subfractions and 1/75th of the product was used for the other four sub-fractions (0–9.5 [#1], 13.5–24 [#2], 24–34.5 [#3] and 34.5–45 min [#4]) (Fig. S2 A). (C) HRMS chromatogram of HexCer (d18:0/16:0) in serum. The colored shadows indicate the peaks coincident with the estimated RT of α-GalCer (d18:0/16:0) (red) and β-GlcCer (d18:0/16:0) (yellow), respectively. (D) HRMS-EIC chromatograms of the synthesized four diastereomers of HexCer (d18:0/16:0) obtained using SFC/HRMS. SFC separated the four diastereomers according to their RTs. The red shadow indicates the peak coincident with the RT of synthesized α-GalCer (d18:0/16:0). (E) HRMS/MS spectra of candidates for α-GalCer (d18:0/16:0) in serum. From top to bottom, collision energy settings are −10 eV (+), −20 eV (++), −30 eV (+++), and −40 eV (++++). The HRMS/MS of precursor-product ion pair: m/z 702.5878 to 540.5350, 522.5245, 284.2948, and 266.2842, respectively. (F) HRMS chromatograms of HexCer (d18:0/16:0) and the HRMS/MS spectra of candidates for α-GalCer (d18:0/16:0) in serum spiked with synthesized α-GalCer (d18:0/16:0). From left to right, collision energy settings are −10 eV (+), −20 eV (++), −30 eV (+++), and −40 eV (++++). The structures of product ions are shown. The red shadow indicates the peak coincident with the RT of synthesized α-GalCer (d18:0/16:0). The mass error tolerance of the precursor ion (m/z 702.5878 as [M+H]+) was <7 ppm (E and F). The HRMS/MS of precursor-product ion pair: m/z 702.5878 to 540.5350, 522.5245, 284.2948, and 266.2842, respectively (E and F). Data are presented as mean ± SD (B) and are representative of three independent experiments (A, C, and D–F).