DIRECT DETERMINATION OF PHOSPHOLIPID STRUCTURES IN MICROORGANISMS BY FAST-ATOM-BOMBARDMENT TRIPLE QUADRUPOLE MASS-SPECTROMETRY

When phospholipids ionized by fast atom bombardment undergo collisionally induced dissociation (CID), they cleave at specific bonds between the functional groups contained on the lipid. These cleavages are common to all classes of phospholipids. By taking advantage of this fact, a general scheme has been developed that uses a triple-quadrupole mass spectrometer to rapidly characterize the phospholipid content and structures present in crude lipid extracts. This scheme is based on fast atom bombardment ionization of a crude lipid extract and on the combination of positive-ion neutral-loss and parent scans and negative-ion daughter scans. Neutral-loss and parent scans provide independent diagnostic mass spectra for each of many specific phospholipid classes, while daughter scans provide the emperical formulas and positions of the fatty acyl constituents on each phospholipid. An automated tandem mass spectrometry (MS/MS) instrument can perform an extensive phospholipid screening on a single sample. A useful mass profile of the phosphatidylethanolamine species present in a 1-pg sample of mixed phospholipids (equivalent to ten Escherichia coli cells) has been obtained. The spectra are reproducible and proportional to concentration over at least the five-logarithm range of cell concentrations studied. A rapid extraction procedure combined with the automated instrument control program produces profiles of the phospholipid classes, along with fatty acyl empirical formulas and position information, on selected phospholipid species, in a few minutes, from a single sample.