Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) in most gram-negative bacteria, and its structure and biosynthetic pathway are well known. Nevertheless, the mechanisms of transport and assembly of this molecule at the cell surface are poorly understood. The inner membrane (IM) transport protein MsbA is responsible for flipping LPS across the IM. Additional components of the LPS transport machinery downstream of MsbA have been identified, including the OM protein complex LptD/LptE (formerly Imp/RlpB), the periplasmic LptA protein, the IM-associated cytoplasmic ATP binding cassette protein LptB, and LptC (formerly YrbK), an essential IM component of the LPS transport machinery characterized in this work. Here we show that depletion of any of the proteins mentioned above leads to common phenotypes, including (i) the presence of abnormal membrane structures in the periplasm, (ii) accumulation of de novo-synthesized LPS in two membrane fractions with lower density than the OM, and (iii) accumulation of a modified LPS, which is ligated to repeating units of colanic acid in the outer leaflet of the IM. Our results suggest that LptA, LptB, LptC, LptD, and LptE operate in the LPS assembly pathway and, together with other as-yet-unidentified components, could be part of a complex devoted to the transport of LPS from the periplasmic surface of the IM to the OM. Moreover, the location of at least one of these five proteins in every cellular compartment suggests a model for how the LPS assembly pathway is organized and ordered in space.

The protein glycosylation is a post-translational modification of crucial importance for its involvement in molecular recognition, protein trafficking, regulation, and inflammation. Indeed, abnormalities in protein glycosylation are correlated with several disease states such as cancer, inflammatory diseases, and congenial disorders. The understanding of cellular mechanisms through the elucidation of glycan composition encourages researchers to find analytical solutions for their detection. Actually, the multiplicity and diversity of glycan structures bond to the proteins, the variations in polarity of the individual saccharide residues, and the poor ionization efficiencies make their detection much trickier than other kinds of biopolymers. An overview of the most prominent techniques based on mass spectrometry (MS) for protein glycosylation (glycoproteomics) studies is here presented. The tricks and pre-treatments of samples are discussed as a crucial step prodromal to the MS analysis to improve the glycan ionization efficiency. Therefore, the different instrumental MS mode is also explored for the qualitative and quantitative analysis of glycopeptides and the glycans structural composition, thus contributing to the elucidation of biological mechanisms.