NACE-ESI-MS/MS method for separation and characterization of phosphorylation and acylation isomers of lipid A.

Lipid A represents a heterogeneous group of bacterial outer membrane phosphoglycolipids, which play a major role in the pathogenesis of Gram-negative sepsis. The number and position of phosphoryl and acyl groups in lipid A molecules are key structural determinants in their bioactivities. In this study, a NACE-ESI-MS/MS method was developed for the simultaneous analysis of lipid A isomers possessing a different degree of phosphorylation and acylation. Various C4'- and C1-monophosphorylated lipid A isobars, as well as acylation isomers, were baseline separated within 43 min in a separation medium of methanol/dichloromethane/triethylamine/acetic acid 60:40:1.08:0.36 (v/v/v/v). Both normal and reverse CE polarities could be applied for proper detection of the analytes owing to the combination of a suction effect caused by the nebulizer gas at the outlet end of the capillary and external pressure applied on the inlet vial. The separated lipid A species could be identified unequivocally by their characteristic fragmentation patterns through CID performed in both negative- and positive-ionization modes. The uniqueness of the NACE-ESI-MS/MS method lies in its simplicity and reliability for proving the phosphorylation isomerism (C1 or C4') and acylation pattern of native lipid A species or those designed for therapeutic applications.

Advanced online mass spectrometry detection of proteins separated by capillary isoelectric focusing after sequential injection.

Capillary isoelectric focusing hyphenated with mass spectrometry detection, following the sequential injection of the carrier ampholytes and the sample zone, is highly efficient for the characterization of proteins. The main advantage of the sequential injection protocol is that ampholytes, with pH ranges, which are not supposed to cover the isoelectric points of the sample components, can be used for separation. The method then allows online mass spectrometry detection of separated analytes either in the absence (substances that have left the pH gradient) or in the presence of low-level ampholytes (substances that are migrating within the pH gradient). The appearance of the substances within, or outside the pH gradient depends on, e.g., the composition of the ampholytes (broad or narrow pH range) or on the composition of electrolyte solutions. The experiments performed in coated capillaries (with polyvinyl alcohol or with polyacrylamide) show that the amount and the injection length of the ampholytes influence the length of the pH gradient formed in the capillary.