Structural characterization of bacterial lipopolysaccharides with mass spectrometry and on‐ and off‐line separation techniques

Abstract

The focus of this review is the application of mass spectrometry to the structural characterization of bacterial lipopolysaccharides (LPSs), also referred to as "endotoxins," because they elicit the strong immune response in infected organisms. Recently, a wide variety of MS-based applications have been implemented to the structure elucidation of LPS. Methodological improvements, as well as on- and off-line separation procedures, proved the versatility of mass spectrometry to study complex LPS mixtures. Special attention is given in the review to the tandem mass spectrometric methods and protocols for the analyses of lipid A, the endotoxic principle of LPS. We compare and evaluate the different ionization techniques (MALDI, ESI) in view of their use in intact R- and S-type LPS and lipid A studies. Methods for sample preparation of LPS prior to mass spectrometric analysis are also described. The direct identification of intrinsic heterogeneities of most intact LPS and lipid A preparations is a particular challenge, for which separation techniques (e.g., TLC, slab-PAGE, CE, GC, HPLC) combined with mass spectrometry are often necessary. A brief summary of these combined methodologies to profile LPS molecular species is provided.

Analysis of Bacterial Lipooligosaccharides by MALDI-TOF MS with Traveling Wave Ion Mobility

“…Very few groups have attempted structural analysis of intact LOS, with or without any preliminary chromatographic separation [24,[28][29][30]. Recently, an improved method was introduced for the analysis of intact LOS by negative-ion, matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) that utilizes a thin layer matrix composed of 2,4,6-trihydroxyacetophenone (THAP) and nitrocellulose [31].…”

“…Recently, an improved method was introduced for the analysis of intact LOS by negative-ion, matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) that utilizes a thin layer matrix composed of 2,4,6-trihydroxyacetophenone (THAP) and nitrocellulose [31]. We and others have exploited this methodology to profile complex LOS mixtures without any chemical modifications [9,10,30,32]. In the MS analysis, LOS molecular ions readily undergo Bprompt^fragmentation, a type of insource decay occurring at the sample surface in a few picoseconds to nanoseconds before or during desorption [33], to give fragments arising from the oligosaccharide and lipid A domains of the molecule through cleavage at the labile ketosidic linkage [31,34].…”

Global and Targeted Lipid Analysis of Gemmata obscuriglobus Reveals the Presence of Lipopolysaccharide, a Signature of the Classical Gram-Negative Outer Membrane

“…Moreover, the inherent chemical heterogeneity of a particular LPS, derived from the interconnection of its three very different regions, makes it challenging to isolate and characterize (33,38,39). Beyond this, structural variations can also occur within the LPS of the same organism (39).…”

“…Moreover, the inherent chemical heterogeneity of a particular LPS, derived from the interconnection of its three very different regions, makes it challenging to isolate and characterize (33,38,39). Beyond this, structural variations can also occur within the LPS of the same organism (39). Regardless of the huge variations possible in LPS structure, the identification of lipid A, its characteristic disaccharide, and 3-HOFAs, and Kdo or Ko, representing the core oligosaccharide, are strong indications for the presence of an LPS in a particular organism.…”

Molecular characterization and expression analysis of purple acid phosphatase gene from pearl oyster Pinctada martensii

“…LPS are large molecules consisting of a lipid and a polysaccharide. These molecules are found in the outer membrane of Gram-negative bacteria and function as endotoxins that elicit a strong immune response in animals (Kilár et al, 2012). PAP expression is significantly upregulated at 48 h after LPS stimulation in human macrophages (Bune et al, 2001).…”