sists of phosphorylated diglucosamines to which from four to seven fatty acids are attached by acyl or amide linkages and forms the anchor to the membrane for the intact LPS. Generally the stoi chiometric quantities of phosphate, glucosamine, and the different fatty ac ids can be established by analysis of hydrolized lipid A, but information about the positions of attachment are best determined by analysis of the frag mentation pattern from the intact mol ecule. In addition, these compounds of ten exhibit considerable heterogeneity. The plasma desorption mass spectra generally show cleavages of the acyllinked fatty acids on the 3- and 3'-positions and formation of an oxonium ion containing only the distal sugar and its fatty acid constituents. The intact LPS, containing lipid A—a core region composed (primarily) of several 2-ketooctanoate (KDO) moieties—and an antigenic portion containing neutral sugars, are a far greater challenge to MS (Figure 8). However, the intact LPS from the rough (Re) mutant of E. coli has a somewhat simpler structure. It has recently been isolated and puri fied by the Takayama group (70). The plasma desorption mass spectrum in Figure 9a was used to confirm the addi tion of two KDO units to the known structure of the lipid A portion. At the Mayo Clinic, Jardine et al. (71) have used PDMS for the structural anal ysis of lipooligosaccharide (LOS) anti gens from Mycobacterium kansasii. FAB mass spectra had been obtained previously for the oligosaccharide por tion of these trehalose-containing lipooligosaccharides, and PDMS spectra of intact LOS revealed the attachment of three 2,4-dimethyltetradecanoyl groups. This group also used PDMS to analyze polysulfated amino glycans (72) and oth er more complex bacterial antigen oligo saccharides, lipooligosaccharides, glycoproteolipids, and larger heparin frag ments (73). Figure 9b shows the mass spectrum of a phenolicglycolipid from M. leprae. Townsend et al. (74) have carried out plasma desorption analyses of proteo lytic glycopeptides from bovine fetuin to elucidate glycosylation site microheterogeneity. Whole cells from bacte ria and intact membranes from rat brain myelin have also been deposited on aluminized mylar foils and analyzed with PDMS by Heller et al. (75). The specific biomarkers phosphatidyl cho line, phosphatidyl ethanolamine, cerebrosides, and sulfatides are observed depending upon species and the charge sign of the phospholipids when ana lyzed by positive and negative PDMS. Summary
The future for PDMS, and for MS in general, is exciting. It is clear that MS will play an important role in biotech-
Figure 8. General structure of the Ra to Re chemotype lipopolysaccharide structure from the rough mutants of gram-negative bacteria. (S = sugar; GIc = glucose; Gal = galactose; GIcNAc = W-acetylglucosamine; Ρ = phosphate; EtN = eth anolamine; KDO = 2-ketooctonate moieties; Ri and R2 are polar head groups.)
nology, and the PDMS technique has already proven to be a sensitive and accurate method for rapid (indeed, routine) comparisons among chemical ly synthesized, recombinant, and na tive peptides. A few years ago, mass spectroscopists would have been sur prised at the possibilities for making such large ions, but we have since ap preciated that the ruggedness of these peptides, as they are transferred from the condensed to the gaseous phase, could not be extrapolated from our pri or experience with molecules with less
defined tertiary structure. At the same time, the remarkable stability of such ions poses a problem in obtaining the usual structural information from ionic fragments. In this regard, attempts to induce fragmentation in high-mass, high-performance (and high-cost) four-sector instruments will be paral leled by the use of chemical and highly specific enzymatic cleavages in situ (i.e., on the PDMS foil or in the FAB matrix), where structural information will be obtained by molecular ion mea surements of the reaction products.
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 13, JULY 1, 1988 · 791 A
