Figure 2.

MPI species from M. tuberculosis H37Rv and selective labeling of the alkyl backbone of C₃₂ MPI. (A) Model 1 is based on sequential condensation of IPP moieties to form a polyisoprenol, which is subsequently acted upon an enzyme with saturase activity. In Model 2, the alkyl backbone is primed by a fatty acyl group and extended by alternating condensation of malonate and methylmalonate in repeated cycles to yield a carboxylic acid product. (B and C) An MPI antigen purified from M. tuberculosis H37Rv was subjected to ESI-MS in the negative mode. The major product, C₃₂ MPI (m/z 707.6), differed in size from the major product from M. avium (m/z 707.6; Table I), and lower abundance ions at m/z 679.6, 693.7, 721.6, and 735.6 were also seen, providing evidence for structural diversity of these natural compounds. Low energy CID-MS/MS for compounds of m/z 679.6, 707.6, and 735.6 generated fragments ions corresponding to a hexosyl phosphate ion (m/z 241) as well as saturated alkyl phosphates corresponding to C₃₀ (m/z 517), C₃₂ (m/z 545), and C₃₄ (m/z 573), respectively, demonstrating that the size variations are accounted for by differences in sizes of the alkyl moiety. (D) High energy CID-MS/MS (double-focusing magnetic sector mass spectrometer) for C₃₂ MPI shows a series of ions at 14-u or 28-u intervals. The 28-u intervals (arrows) correspond to methyl branches at C₄, C₈, C₁₂, C₁₆, and C₂₀. (E) M. tuberculosis H37Rv was grown in the presence of ¹³C-labeled propionate or nonlabeled propionate and subjected to lipid extraction and analysis using a quadruple ion trap mass spectrometer. Compared with unlabeled MPI, the mass shift of the main product from m/z 707.6 to 713.6 corresponded to the incorporation of six C₃ units in each molecule. MS/MS spectra of m/z 713.6 shows that the alkyl phosphate fragment (m/z 551.5) was shifted by six mass units compared with the corresponding fragment from unlabeled C₃₂ MPI (m/z 545.5; panel C), indicating that as many as six C₃ units are incorporated into the alkyl chain.