Terminal Galactosylation and Sialylation Switching on Membrane Glycoproteins upon TNF-Alpha-Induced Insulin Resistance in Adipocytes

Abstract

Insulin resistance (IR) is a complex pathophysiological state that arises from both environmental and genetic perturbations and leads to a variety of diseases, including type-2 diabetes (T2D). Obesity is associated with enhanced adipose tissue inflammation, which may play a role in disease progression. Inflammation modulates protein glycosylation in a variety of cell types, and this has been associated with biological dysregulation. Here, we have examined the effects of an inflammatory insult on protein glycosylation in adipocytes. We performed quantitative N-glycome profiling of membrane proteins derived from mouse 3T3-L1 adipocytes that had been incubated with or without the proinflammatory cytokine TNF-alpha to induce IR. We identified the regulation of specific terminal N-glycan epitopes, including an increase in terminal di-galactose-and a decrease in biantennary alpha-2,3-sialoglycans

Maturing Glycoproteomics Technologies Provide Unique Structural Insights into the N-glycoproteome and Its Regulation in Health and Disease

“…Some glycoproteomics approaches even by-pass this step because of the increased capacity of modern LC-MS/MS instrumentation to handle the extreme complexity of biologically-relevant glycopeptide mixtures, and perhaps because the multiple LC fractions resulting from off-line separations dramatically increase the required LC-MS/MS instrument time, lower the overall sensitivity of the analysis and complicate the downstream quantitative data analysis (103,104). However, multiple glycoproteomics strategies, particularly in studies where the amounts of biological sample material are not a limiting factor, still opt to use off-line separation to increase the glycoproteome coverage (46,(105)(106)(107). The most exciting new developments in this area include the off-line use of neutral/high pH (pH 7-10) RP-LC (7,108), which displayed orthogonal glycopeptide retention behavior relative to the conventional acidic (ϳpH 2) RP-LC-(online) MS/MS.…”

Structural characterisation of glycosylation of paramyxovirus attachment and fusion proteins by mass spectrometry

“…The panel has a schematic of the peptide fragmentation pattern observed for the glycopeptide (hRSV-G aa [222][223][224][225][226][227][228][229][230][231][232][233][234][235][236][237][238][239][240]. Not all ions have been labelled on the spectrum for ease of interpretation.…”

“…Enrichment is particularly relevant for complex samples and, depending on the approach, can facilitate the isolation of a broad range of glycopeptides (206,213,227,230) or those carrying certain monosaccharides or substituents (235)(236)(237)(238)(239). Hydrophilic interaction liquid chromatography is commonly reported in the literature (234) with several different types of resins and methods available (240)(241)(242).…”

“…Chapter 1: Introduction to the structure and function of glycosylation with reference to paramyxovirus surface proteins 23 (224)(225)(226) with ETD, (termed EThcD or ETciD when combined with HCD or CID, respectively) has been used to enhance fragmentation of N-linked (227) and O-linked glycopeptides (221). In addition to increasing the relative amounts of c-and z-ions these combined fragmentation methods also produce ions that are characteristic of CID/HCD fragmentation of glycopeptides (19).…”

“…These strategies used high mass accuracy glycan oxonium ions detected in the first pass Orbitrap HCD scans to trigger 'on-the-fly' ETD or CID fragmentation of the same precursor ion detected in the Orbitrap or ion trap, respectively (227)(228)(229)(230)(231). These strategies allow targeted sampling of glycopeptides by complementary fragmentation techniques in one MS run without a drastic increase in duty cycle, which is the time taken to monitor an analyte.…”