Diversity of non‐stoichiometric substitutions on the lipopolysaccharide of E. coli C demonstrated by electrospray ionization single quadrupole mass spectrometry

Abstract

The lipopolysaccharide (LPS) of enterobacteria frequently contains various numbers of charged non-stoichiometric substituents such as phosphate (P) and ethanolamine (EtN) groups and a third residue of 3-deoxy-D-manno-2-octulosonic acid (KDO) on the R-core polysaccharide backbone. These substituents can modify the biological activities of LPS including varying the stability of the outer membrane, tolerance to cationic antibiotics, pathogenicity, and sensitivity to enterobacteria bacteriophages. These diverse substituents can be clearly detected in degraded samples of LPS from E. coli C using electrospray ionization single quadrupole mass spectrometry (ESI-Q-MS) from a 0.1 mg/mL solution in a 50:50 mixture of methanol and 10 mM ammonium acetate (pH 6.8). The O-deacylated derivative showed multiple peaks of [M-3H](3-) ions which corresponded to species having up to eight phosphates, two ethanolamines, and an additional KDO on the backbone of Hex(5) Hep(3) KDO(2) GlcN(2) C14:0(3-OH)(2). The major components of the O,N-deacylated derivative were the species associated with four and five phosphates on Hex(5) Hep(3) KDO(2) GlcN(2). The polysaccharide portion of LPS also revealed species which corresponded to Hex(5) Hep(3) KDO associated with two to four phosphates and an ethanolamine. The present method was proved to be useful to investigate the structural diversity of enterobacterial LPS.

Improved separation and characterization of lipopolysaccharide related compounds by reverse phase ion pairing-HPLC/electrospray ionization-quadrupole-mass spectrometry (RPIP-HPLC/ESI-Q-MS)

“…In addition, peaks corresponding to the dehydration products were also observed. These dehydration products were detected in our previous study of the ESI-Q-MS analysis of the purified PS derivative [21]. In that study it was not clear whether these were generated during the acid hydrolysis of LPS or during the MS measurement.…”

“…However, the number and position of the non-stoichiometric substituents are variable and depend on the growing conditions. Previous studies have successfully demonstrated electrospray ionization-mass spectrometry (ESI-MS) can determine the diversity of such non-stoichiometric substitutions on LPS related compounds, such as O-deacylated (deO), O,N-deacylated (deON), and polysaccharide portion (PS), derived from the limited chemical degradation of the LPS [21]. The multiple species were detected for deON and PS derivatives by use of strongly basic anion exchange HPLC with post-column fluorescence derivatization (SAX-HPLC/FLD) and capillary zone electrophoresis with electrospray ionization ion-trap mass spectrometry (CZE/ESI-IT-MS) [6].…”

Liquid Chromatography/Electrospray Ionization Tandem Mass Spectrometry‐based Structural Analysis of Deacylated Lipooligosaccharides From Escherichia coli

“…Over the past few years, mass spectrometry, especially matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS), has been widely used for structural analysis of high molecular weight compounds due to its specific advantage in characterizing them . The structural analysis of intact LPS‐derivatives by MS is very difficult, while parts of lipid A species and oligosaccharides can be analyzed by MS/MS . However, the method of analyzing LPS‐derivatives using MALDI‐TOF shows low analytical reproducibility due to the low ionization efficiency of LPS‐derivatives.…”

Shotgun Analysis of Rough-Type Lipopolysaccharides Using Ultraviolet Photodissociation Mass Spectrometry

“…10,18,19 Additionally, lipid A acyl chains can be removed by hydrolysis prior to analysis of the intact carbohydrate chain. Collisional induced dissociation (CID) is commonly used to obtain fragmentation patterns for the hydrolyzed lipid A 20–25 and saccharide 26 substructures. However, CID does not always provide adequate fragmentation for elucidation of subtle structural differences.…”

“…26,30,31 Recently, we examined the fragmentation patterns of intact R-LPS using a hybrid UVPD/HCD method. 32 UVPD and hybrid UVPD/HCD produced very rich fragmentation patterns arising from a diverse array of C-O, C-N, and C-C cleavages from both the lipid and oligosaccharide portions of the LPS, as illustrated for endotoxin structures from E. coli .…”