Figure 3.

Negative ion ESI/MS analysis of total F. novicida lipids and of the purified GalN donor. A. The spectrum of the total lipids from F. novicida strain XWK2 (15) reveals major peaks between m/z 500-900, arising mainly from glycerophospholipids. The peaks near m/z 1476 and 1504 are attributed to free lipid A; this strain lacks the usual GalN substituent because of an insertion mutation in the GalN transferase flmK(arnT) (15). However, the spectrum of wild-type F. novicida U112 lipids is virtually identical, except that the free lipid A is seen near m/z 1637 and 1655 (not shown) (15). Inset. The [M-H]^- ion of the proposed undecaprenyl phosphate-α-d-GalN (Fig. 2) donor substrate is predicted at m/z 1006.726, and a peak is observed at m/z 1006.698. However, an additional overlapping peak, probably arising from an undecaprenyl phosphate-glucose, is seen at m/z 1007.603, partially obscuring the undecaprenyl phosphate-α-d-GalN isotopic peak containing one ¹³C atom. B. The spectrum of the purified GalN donor lipid shows a major peak, interpreted as [M-H]^-, at m/z 1006.699. Inset. The overlapping putative undecaprenyl phosphate-hexose [M-H]^- peak at m/z 1007.603 seen in panel A was removed by the purification; what remains at m/z 1007.705 can now be attributed to an undecaprenyl phosphate-α-d-GalN isotopic species containing one ¹³C atom. The smaller peak at m/z 1074.764 is consistent with the [M-H]^- of a dodecaprenyl phosphate-GalN derivative. The peak at m/z 1022.698 might arise by chemical oxidation of undecaprenyl phosphate-α-d-GalN generated during TLC.