Detection of α-GalCer (d18:0/16:0) in mammalian tissues. (A) HRMS/MS spectra of candidate for α-GalCer (d18:0/16:0) (m/z 702.5878, as [M+H]+) in mouse spleen (left) and thymus (right). All HRMS/MS mass error tolerances are <7 ppm. From top to bottom, collision energy settings are −10 eV (+), −20 eV (++), −30 eV (+++), and −40 eV (++++). (B and C) HRMS chromatograms of HexCer (d18:0/16:0) and the HRMS/MS spectra of candidates for α-GalCer (d18:0/16:0) in spleen (B) and thymus (C) spiked with synthesized α-GalCer (d18:0/16:0). The area under the curve value ratios of each peak coincident with synthesized α-GalCer (d18:0/16:0) are 3.67 × 105:7.35 × 105 (B) and 3.43 × 105:5.94 × 105 (C). The mass error tolerance of the precursor ion (m/z 702.5878 as [M+H]+) was <7 ppm (A–C). The HRMS/MS chromatograms of precursor-product ion pair: m/z 702.5878 to 540.5350, 522.5245, 284.2948, and 266.2842, respectively (A–C). The red shadow indicates the peak coincident with synthesized α-GalCer (d18:0/16:0) (B and C). From left to right, collision energy settings are −10 eV (+), −20 eV (++), −30 eV (+++), and −40 eV (++++) (B and C). The structures of product ions are shown (B and C). Data are representative of three independent experiments (A–C).
Figure 4.

Detection of α-GalCer (d18:0/16:0) in mammalian tissues. (A) HRMS/MS spectra of candidate for α-GalCer (d18:0/16:0) (m/z 702.5878, as [M+H]+) in mouse spleen (left) and thymus (right). All HRMS/MS mass error tolerances are <7 ppm. From top to bottom, collision energy settings are −10 eV (+), −20 eV (++), −30 eV (+++), and −40 eV (++++). (B and C) HRMS chromatograms of HexCer (d18:0/16:0) and the HRMS/MS spectra of candidates for α-GalCer (d18:0/16:0) in spleen (B) and thymus (C) spiked with synthesized α-GalCer (d18:0/16:0). The area under the curve value ratios of each peak coincident with synthesized α-GalCer (d18:0/16:0) are 3.67 × 105:7.35 × 105 (B) and 3.43 × 105:5.94 × 105 (C). The mass error tolerance of the precursor ion (m/z 702.5878 as [M+H]+) was <7 ppm (A–C). The HRMS/MS chromatograms of precursor-product ion pair: m/z 702.5878 to 540.5350, 522.5245, 284.2948, and 266.2842, respectively (A–C). The red shadow indicates the peak coincident with synthesized α-GalCer (d18:0/16:0) (B and C). From left to right, collision energy settings are −10 eV (+), −20 eV (++), −30 eV (+++), and −40 eV (++++) (B and C). The structures of product ions are shown (B and C). Data are representative of three independent experiments (A–C).

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