Characterization of complex, heterogeneous lipid A samples using HPLC‐MS/MS technique III. Positive‐ion mode tandem mass spectrometry to reveal phosphorylation and acylation patterns of lipid A

Abstract

In this study, we report the detailed analysis of the fragmentation patterns of positively charged lipid A species based on their tandem mass spectra obtained under low-energy collision-induced dissociation conditions of an electrospray quadrupole time-of-flight mass spectrometer. The tandem mass spectrometry experiments were performed after the separation of the compounds with a reversed-phase high performance liquid chromatography method. We found that both, phosphorylated and nonphosphorylated lipid A molecules can be readily ionized in the positive-ion mode by adduct formation with triethylamine added to the eluent. The tandem mass spectra of the lipid A triethylammonium adduct ions showed several product ions corresponding to inter-ring glycosidic cleavages of the sugar residues, as well as consecutive and competitive eliminations of fatty acids, phosphoric acid, and water following the neutral loss of triethylamine. Characteristic product ions provided direct information on the phosphorylation site(s), also when phosphorylation isomers (ie, containing either a C1 or a C4' phosphate group) were simultaneously present in the sample. Continuous series of high-abundance B-type and low-abundance Y-type inter-ring fragment ions were indicative of the fatty acyl distribution between the nonreducing and reducing ends of the lipid A backbone. The previously reported lipid A structures of Proteus morganii O34 and Escherichia coli O111 bacteria were used as standards. Although, the fragmentation pathways of the differently phosphorylated lipid A species significantly differed in the negative-ion mode, they were very similar in the positive-ion mode. The complementary use of positive-ion and negative-ion mode tandem mass spectrometry was found to be essential for the full structural characterization of the C1-monophosphorylated lipid A species.

Phosphoglycolipid Profiling of Bacterial Endotoxins

“…Based on previously published data on the acyl distribution of the C4'-monophosphorylated, hexaacylated lipid A molecule from E. coli O111 [6], ions observed in the tandem mass spectra (not shown) of this lipid A species (appearing at t R = 29.2 mins in Fig. 2) could be structurally assigned in both ionization modes [3,5]. Using this as a standard, the CID mass spectral profiles of other monophosphorylated lipid A species separated by chromatography were compared, and the fragmentation preferences for each of them, in both ionization modes, were explored.…”

“…The main advantage of using the collision cell of a Q-TOF mass spectrometer was that several generations of precursor/product ions -that would otherwise be generated only at higher MS stages with ion-trap experiments -were observed simultaneously in the tandem mass spectra of the separated lipid A species. This facilitated new fragmentation rules to be set for the variety of phosphorylated and acylated lipid A compounds by a simple MS/MS experiment [3][4][5]. Specifically, the acylation profile of the non-, C4'-mono and bisphosphorylated lipid A species could be inferred partly from the positiveand fully from the negative-ion mode MS/MS analyses [3][4][5], whereas the complementary use of both ionization modes [3,5] was needed for the full structural characterization of C1-monophosphorylated lipid A.…”

“…Next, the linkages at the 2 ε (only from the Btype ion) and 2ε positions can be determined from the positive-ion mode analysis. By knowing the fragmentation preference of the secondary ester bonds (which is 2 ε > 3 ε > 2ε [5]), substitutions at these sites can be deconvoluted from the negative-ion mode analysis by observing the 2 ε, 3 ε, 2ε, and 3α cleavage products in the upper m/z region and those resulting from the 3 α, 2 ε + 3 ε, 2 ε + 2ε, 2 ε + 3α, and 3 ε + 3α cleavages displayed in the middle m/z region. Here, the dephosphorylated Z-type ion in the lower m/z region indirectly specifies the linkage at the 2α position.…”

“…The fragmentation behavior of non-and bisphosphorylated lipid A species under both negative and positive CID conditions was investigated, as well. For these, a different sequencing of the acyl-chain cleavages could be observed in the negative-ion mode [3,4], whereas similar fragmentation pathways to that of the monophosphorylated species in the positive-ion mode were identified [5]. Besides the A-type ions (nonreducing end), complementary X-type fragment ions (reducing end) appeared in the negative-ion mode tandem mass…”

“…Our method is based on a reversedphase high-performance liquid chromatography (RP-HPLC) separation and online ESI-Q-TOF mass spectrometric detection of molecular species in native lipid A isolates. The exact structures of the lipid A molecules in the complex mixtures were deduced from specific fragmentation patterns obtained in both the negative- [3,4] and positive-ion modes [5].…”