Lipopolysaccharide (LPS) and Protein-LPS complexes: Detection and Characterization by Gel Electrophoresis, Mass Spectrometry and Bioassays

Abstract

A prerequisite to the discovery and characterization of lipopolysaccharide (LPS) interaction with specific receptors and the resulting pathophysiological effects is the comprehensive structural analysis of LPS species. This brief review is aimed to summarize the use of gel electrophoresis linked to other biochemical technologies for detecting and characterizing LPSs. Lipopolysaccharide aggregates alone or mixtures containing LPSs and proteins/peptides can be separated by native agarose gel electrophoresis (NAGE), after which LPSs are detected with imidazole and zinc salts. A double-staining process with Coomassie brilliant blue R-250 enables the use of NAGE for detecting and studying protein-LPS interactions. For compositional analysis, the LPS aggregates are separated, with high resolution, by surfactant-polyacrylamide gel electrophoresis. After reverse staining with zinc-imidazole and elution from gel microparticles, glycoform-specific LPSs are ready for structural and biological analysis. For sequence analysis based on tandem electrospray ionization mass spectrometry (ESI-MS/MS) of oligosaccharides, LPSs are subjected to mild acid hydrolysis, dephosphorylation and permethylation. Also, O-deacylated LPS forms can be analyzed by matrix-assisted laser desorption/ionization-time of flight-MS. By comparison to spectra of unpurified LPSs, mass spectra of the micropurified LPSs show reduce heterogeneity and increased signal-to-noise ratios. Furthermore, the micropurification of LPSs prior to MS allows a higher sensitivity of detection for less abundant LPS glycoforms. The micropurified LPS fractions can be used to form self-assembled nanoaggregates which may be detected by dynamic light scattering. The effect of the O-side chain length on the Z-potential of LPS aggregates may be estimated by measurements based on laser Doppler electrophoresis. The thus obtained glycoform-specific LPSs are not only intact chemically but also biologically active as tested by e.g. Limulus amoebocyte lysate test, TNF-α assay and agonistic effect on human Toll-like receptor 4.

Regenerative Core–Shell Nanoparticles for Simultaneous Removal and Detection of Endotoxins

“…13 To this, we further added SDS or EDTA to ensure that the LPS micelles were fully broken into monomers. 33,34 This led to a 1.5% increase in fluorescence intensity in the presence of EDTA and 1.25% with SDS (in comparison to buffer alone) (Figure S4B). The LPS-capturing efficiency achieved was, however, only 16%.…”

Exploring Humic Acid as an Efficient and Selective Adsorbent for Lead Removal in Multi-Metal Coexistence Systems: A Review

“…Additionally, the absorption intensity of aliphatic and amide groups increased, while the carboxyl group intensity decreased. Jovanovic et al [41] explored the pH (2-11) dependency of HA particle size, finding that HAs exhibit large molecular aggregates or supramolecular structures. Klučáková et al [42] investigated the linear relationship between functional group intensity and the dissociation ability of HAs at different pH levels.…”

Stability and Activity of Lipid a Phosphoethanolamine Transferase From Neisseria Meningitidis in the Presence of Sodium Deoxycholate [Stabilitas Dan Aktivitas Lipid a Phosphoethanolamine Transferase Dari Neisseria Meningitidis Terhadap Penambahan Natrium Deoksikolat]

“…To study the structure and catalytic mechanism of EptA, particularly in the presence of its substrates (PE and LOS), another surfactant namely sodium deoxycholate (DOC), is needed in order to prevent aggregation of LPS/LOS (Komuro & Galanos, 1988& Hardy et al, 2016. DOC is an ionic detergent which tends to denature protein thus, in this research the effect of DOC on the activity and structural stability of EptA was investigated.…”