Fig 1.
P. furiosus contains heptasaccharide-charged DolP.
A. The mass spectrum of the RPLC fraction with a retention time of 8.5–9.5 min contains [M-2H]2- peaks of m/z 1046.592, 1081.120 and 1114.637, corresponding to heptasaccharide-charged C60, C65 and C70 DolP, respectively. The 13C1-containing isotopic and monoisotopic [M-2H]2- peaks of heptasaccharide-charged C65 DolP are indicated. The inset shows [M-2H]2- peaks of m/z 914.551, 948.575 and 979.583, corresponding to C60, C65 and C70 DolP attached to the pentasaccharide precursor of the complete heptasaccharide, as well as [M-2H]2- peaks of m/z 980.585, 1014.089 and 1048.116, corresponding to C60, C65 and C70 DolP attached to the hexasaccharide precursor of the same heptasaccharide. B. The chemical structure (based on the N-linked glycan) and MS/MS fragmentation scheme are shown in the panel top. The MS/MS spectrum of the [M-2H]2- peak of m/z 1081.12 corresponding to heptasaccharide-charged C65 DolP is presented in the panel bottom. The arrows indicating x10 and x15 reflect magnification of the ion peaks in the corresponding region of m/z values on the spectrum.
Fig 2.
The glycan-charged DolP species detected in the P. furiosus lipid extract.
The predicted chemical structures and calculated masses of C65 DolP charged with the mono-, di-, tri-, tetra-, penta-, hexasaccharide-, and the complete heptasaccharide are shown (top to bottom, respectively).
Fig 3.
P. furiosus DolP is highly saturated.
A. The MS spectrum of the NPLC fraction with a retention time of 14–14.5 min contains [M-H]- ion peaks corresponding to N-acetylhexosamine-linked C6o, C65 and C70 DolP (m/z 1126.871, 1194.934 and 1262.995, respectively). Each DolP species includes 7–11 unsaturated isoprene subunits. B. Zoomed-in spectrum of N-acetylhexosamine-charged C65 DolP [M-H]- ion peaks showing the varying degree of dolichol saturation. Peaks corresponding to species containing dolichol that includes 2–6 saturated bonds are indicated accordingly. C. MS/MS spectrum of the [M-H]- ion of C65 DolP at m/z 1194.934. The fragmentation scheme (inset) shows the distribution of the saturated bonds (mostly near the ω-terminus).