Mass spectrometric studies are now playing a leading role in the elucidation of lipopolysaccharide (LPS) structures through the characterization of antigenic polysaccharides, core oligosaccharides and lipid A components including LPS genetic modifications. The conventional MS and MS/MS analyses together with CID fragmentation provide additional structural information complementary to the previous analytical experiments, and thus contribute to an integrated strategy for the simultaneous characterization and correct sequencing of the carbohydrate moiety.

The molecular structure of the mutant form of the lipopolysaccharide of Aeromonas salmonicida was determined to contain an O-4 phosphorylated and O-5 substituted Kdo reducing group, and is proposed as the following: [molecular structure: see text] It was established that during the cleavage of this LPS with 1% acetic acid, to release the core oligosaccharide from the Lipid A portion, we obtained a degraded core oligosaccharide which eliminated its phosphate group with extreme facility. The precise molecular structure of this dephosphorylated core was deduced by electrospray mass spectrometry and is proposed as the following:[molecular structure: see text] Low energy collision ESI-QqTOF-MS/MS analysis of the dephosphorylated core oligosaccharide confirmed the presence of the O-5 glycosylated 4,8- and 4,7-anhydro derivatives of the enolizable alpha-keto-acids. The CID tandem mass spectrometric analysis of the heterogeneous mixture of the permethylated core oligosaccharide established the unreported methylation reaction on the diastereomeric 4,8- and 4,7-anhydro alpha-keto-acids and the complete permethylation and addition reaction of the O-5 glycosylated open chain reducing end terminal D-arabino-3-en-2-ulonic acid. The stereo-specific fragmentation routes obtained during the tandem mass spectrometric analysis permitted the precise sequencing of this dephosphorylated rough core oligosaccharide of the mutant LPS of A. salmonicida.

The molecular structure of the wild strain of the lipopolysaccharide (LPS) core of Aeromonas salmonicida, ssp salmonicida has been sequenced using tandem mass spectrometry. The core oligosaccharide was determined to contain an O-4 phosphorylated and O-5 substituted Kdo reducing group and its structure is proposed as follows:After the core oligosaccharide of LPS was released from the lipid A portion by conventional treatment with 1% acetic acid, we demonstrated the existence of a homogeneous mixture composed mainly of the native core oligosaccharide containing the Kdo with its O-4 phosphate group intact and a degraded core oligosaccharide mixture which eliminated the O-4 phosphate group with extreme ease. The precise molecular structure and glycone sequence of the homogeneous mixture of phosphorylated and dephosphorylated core oligosaccharides was determined by electrospray ionization mass spectrometry and tandem mass spectrometric (MS/MS) analysis. Collision-induced dissociation MS/MS of the homogeneous mixture of permethylated core oligosaccharides afforded a series of diagnostic product ions which confirmed the established sequence of the glycones to be determined. Matrix-assisted laser desorption/ionization MS/MS reconfirmed the molecular structure of the dephosphorylated homogeneous permethylated mixture of the core oligosaccharides containing the diastereomeric 4,8-and 4,7-anhydro-α-keto acids.Keywords: structural investigation, Aeromonas salmonicida ssp salmonicida, lipopolysaccharide, native phosphorylated core oligosaccharide, degraded dephosphorylated core oligosaccharide, electrospray and matrix-assisted laser desorption/ionization, mass spectrometry and tandem mass spectrometry L-α-D-Hepp

The original article to which this Addendum refers was published in Rapid Commun. Mass Spectrom. 2005; 19: 3119–3130.

In this communication, the structural analysis of six synthetic O-Linked amphiphilic cholesteryl polyethoxy neoglycolipids containing N-acetyl-D-glucosamine was performed by electrospray ionization mass spectrometry in the positive ion mode, with a QqTOF-MS/MS hybrid instrument. The MS/MS analyses provided evidence for the "in situ" formation, in the collision cell of the tandem mass spectrometer, of an unexpected and unique [C-glycoside] ϩ product ion, resulting from an ion-molecule reaction between the N-acetyl-D-glucosamine oxonium ion and the neutral cholesta-3,5-diene molecule. Quasi T he structural diversity of complex carbohydrates of cell surface membranes has been much more appreciated over the last two decades, due to the vast improvement in analytical techniques.We have synthesized a series of amphiphilic neoglycolipid cholesteryl derivatives (see Scheme 1), in which the cholesterol and carbohydrate (N-acetyl-D-glucosamine) moieties were attached by means of a polyethoxy variable spacer [1,2]. Synthetic neoglycolipids have been successfully incorporated into liposomal formulations to prolong their half lives as alternative to pegylated liposomes (PEG-liposomes) [3,4]. More recently, novel series of neoglycolipids bearing various sugar monomers or oligomers have been evaluated as stabilizing agents for cationic liposomes [5] which are one of the most important nonviral gene carriers used in cancer gene therapy [6]. In this study, neoglycolipid-stabilized liposomes were superior to PEG-stabilized liposomes in terms of gene transfer efficiency [5].In the present communication, we report the "in situ" formation, both in the collision cell and the ESI interface of the tandem mass spectrometer, of an unexpected and unique [C-glycoside] ϩ product ion, resulting from an ion-molecule reaction between the N-acetyl-D-glucosamine oxonium ion and the neutral cholesta-3,5-diene molecule. We also propose the fragmentation routes of these synthetic amphiphilic cholesteryl polyethoxy neoglycolipids by electrospray ionization mass spectrometry with a QqTOF-MS hybrid instrument. The low-energy collision induced dissociation (CID) tandem mass spectrometric analyses are also described.