Determination of pyrophosphorylated forms of lipid A in Gram-negative bacteria using a multivaried mass spectrometric approach

Abstract

Lipid A isolated from several bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, and various strains of Yersinia) showed abundant formation of pyrophosphate anions upon ion dissociation. Pyrophosphate [H(3)P(2)O(7)](-) and/or [HP(2)O(6)](-) anions were observed as dominant fragments from diphosphorylated lipid A anions regardless of the ionization mode (matrix-assisted laser desorption ionization or electrospray ionization), excitation mode (collisional activation or infrared photoexcitation), or mass analyzer (time-of-flight/time-of-flight, tandem quadrupole, Fourier transform-ion cyclotron resonance mass spectrometry). Dissociations of anions from model lipid phosphate, pyrophosphate, and hexose diphosphates confirmed that pyrophosphate fragments were formed abundantly only in the presence of an intact pyrophosphate group in the analyte molecule and were not due to intramolecular rearrangement upon ionization, ion-molecule reactions, or rearrangement following activation. This indicated that pyrophosphate groups are present in diphosphorylated lipid A from a variety of Gram-negative bacteria.

Fig. 1.

Negative ion mode MALDI-TOF mass spectrum of lipid A from Yp grown at 37°C. (Inset) Structure representing a previously proposed structure for m/z 1,404 (, –18). Shorthand notation is as follows: LA, lipid A; subscript, number of acyl chains and, unless otherwise noted, acyl chains are 3-hydroxymyristic acid with the exception of C12 (lauric acid) and C16:1 (palmitoleic acid); HPO₃, phosphate group; Ara4N, aminoarabinose.

Fig. 2.

Negative ion mode MALDI-TOF/TOF MS² spectrum of m/z 1,404 of lipid A from Yp grown at 37°C (A) and negative ion mode ESI LTQ-FT IRMPD MS² spectrum of m/z 1,404 of lipid A from Yp grown at 37°C (B).

Scheme 1.

Dissociation of m/z 1,404 rationalized by a pyrophosphorylated lipid A structure. Note that although the pyrophosphate moiety is shown at the 1-position, it could have been placed at the 4′-position. The structure of m/z 1,404 is a heterogeneous mixture of bisphosphate and pyrophosphate, and therefore the structure in this scheme is not the only conceivable phosphorylation arrangement.

Fig. 3.

Negative ion mode ESI LTQ-FT mass spectrum of lipid A from Yp grown at 37°C. Refer to Fig. 1 for shorthand notation. All ions are singly deprotonated unless otherwise noted.

Fig. 4.

Negative ion mode ESI LTQ-FT CID MS² spectrum of m/z 1,404 of lipid A from Yp grown at 37°C (A), ESI LTQ CID MS³ of m/z 772 derived from m/z 1,404 (B), and ESI LTQ CID MS⁴ of m/z 528 derived from m/z 772 and 1,404 (C). Structures for m/z 1,404, 772, and 528 ions can be found in Scheme 1.