The cell membrane plays an important role in protecting the cell from its extracellular environment. As such, extensive work has been devoted to studying its structure and function. Crucial intercellular processes, such as signal transduction and immune protection, are mediated by cell surface glycosylation, which is comprised of large biomolecules, including glycoproteins and glycosphingolipids. Because perturbations in glycosylation could result in dysfunction of cells and are related to diseases, the analysis of surface glycosylation is critical for understanding pathogenic mechanisms and can further lead to biomarker discovery. Different mass spectrometry-based techniques have been developed for glycan analysis, ranging from highly specific, targeted approaches to more comprehensive profiling studies. In this review, we summarized the work conducted for extensive analysis of cell membrane glycosylation, particularly those employing liquid chromatography with mass spectrometry (LC-MS) in combination with various sample preparation techniques.

Saccharides in our diet are major sources of carbon for the formation of biomass such as proteins, lipids, nucleic acids and glycans. Among the dietary monosaccharides, glucose occupies a central role in metabolism, but human blood contains regulated levels of other monosaccharides as well. Their influence on metabolism and how they are utilized have not been explored thoroughly. Applying metabolic flux analysis on glycan synthesis can reveal the pathways that supply glycosylation precursors and provide a snapshot of the metabolic state of the cell. In this study, we traced the incorporation of six 13C uniformly labeled monosaccharides in the N-glycans, O-glycans and glycosphingolipids of both pluripotent and neural NTERA-2 cells. We gathered detailed isotopologue data for hundreds of glycoconjugates using mass spectrometry methods. The contributions of de novo synthesis and direct incorporation pathways for glucose, mannose, fructose, galactose, N-acetylglucosamine and fucose were determined based on their isotope incorporation. Co-feeding studies revealed that fructose incorporation is drastically decreased by the presence of glucose, while mannose and galactose were much less affected. Furthermore, increased sialylation slowed down the turnover of glycans, but fucosylation attenuated this effect. Our results demonstrated that exogenous monosaccharide utilization can vary markedly depending on the cell differentiation state and monosaccharide availability, and that the incorporation of carbons can also differ among different glycan structures. We contend that the analysis of metabolic isotope labeling of glycans can yield new insights about cell metabolism.

Glycosphingolipids (GSLs) are found in cellular membranes of most organisms and play important roles in cell-cell recognition, signaling, growth, and adhesion, among others. A method based on nanoflow high performance liquid chromatography-chip-quadrupole-time-of-flight mass spectrometry (nanoHPLC Chip-Q-TOF MS) was applied towards identifying and quantifying intact GSLs from a variety of samples, including cultured cell lines and animal tissue. The method provides the composition and sequence of the glycan, as well as variations in the ceramide portion of the GSL. It was used to profile the changes in the glycolipidome of Caco-2 cells as they undergo differentiation. A total of 226 unique GSLs were found among Caco-2 samples from five differentiation time-points. The method provided a comprehensive glycolipidomic profile of a cell during differentiation to yield the dynamic variation of intact GSL structures.

Cell membrane protein glycosylation is dependent on the metabolic state of the cell as well as exogenous nutrients available. Although the metabolism and interconversion of monosaccharides have been well-studied, their incorporation into cell surface glycans and their corresponding glycoproteins remains relatively unknown. In this study, we developed a method to investigate quantitatively the incorporation pathways of dietary saccharides into specific glycans and glycoproteins on the cell membrane by treating intestinal Caco-2 and hepatic KKU-M213 cells with ¹³C-labeled monosaccharides and characterizing the resulting cell surface glycans and glycopeptides by LC-MS/MS. Time-course studies using uniformly labeled glucose revealed that the rate of incorporation was both glycan-specific and protein-dependent. Comparative studies using different dietary saccharides and multiple cell lines revealed the variance of monosaccharide utilization and interconversion in different tissues and organisms. The robust isotope-labeling and glycan profiling methods can provide a useful tool for differentiating glycosylation pathways and enhance the understanding of how dietary sugar intake affects health.

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Epithelial cells in the lining of the intestines play critical roles in maintaining homeostasis while challenged by dynamic and sudden changes in luminal contents. Given the high density of glycosylation that encompasses their extracellular surface, environmental changes may lead to extensive reorganization of membrane-associated glycans. However, neither the molecular details nor the consequences of conditional glycan changes are well understood. Here we assessed the sensitivity of Caco-2 and HT-29 membrane N-glycosylation to variations in (i) dietary elements, (ii) microbial fermentation products and (iii) cell culture parameters relevant to intestinal epithelial cell growth and survival. Based on global LC-MS glycomic and statistical analyses, the resulting glycan expression changes were systematic, dependent upon the conditions of each controlled environment. Exposure to short chain fatty acids produced significant increases in fucosylation while further acidification promoted hypersialylation. Notably, among all conditions, increases of high mannose type glycans were identified as a major response when extracellular fructose, galactose and glutamine were independently elevated. To examine the functional consequences of this discrete shift in the displayed glycome, we applied a chemical inhibitor of the glycan processing mannosidase, globally intensifying high mannose expression. The data reveal that upregulation of high mannose glycosylation has detrimental effects on basic intestinal epithelium functions by altering permeability, host-microbe associations and membrane protein activities.

The proteins in the cell membrane of the brain are modified by glycans in highly interactive regions. The glycans and glycoproteins are involved in cell-cell interactions that are of fundamental importance to the brain. In this study, the comprehensive N-glycome and N-glycoproteome of the brain were determined in 11 functional brain regions, some of them known to be affected with the progression of Alzheimer's disease. N-glycans throughout the regions were generally highly branched and highly sialofucosylated. Regional variations were also found with regard to the glycan types including high mannose and complex-type structures. Glycoproteomic analysis identified the proteins that differed in glycosylation in the various regions. To obtain the broader representation of glycan compositions, four subjects with two in their 70s and two in their 90s representing two Alzheimer's disease subjects, one hippocampal sclerosis subject, and one subject with no cognitive impairment were analyzed. The four subjects were all glycomically mapped across 11 brain regions. Marked differences in the glycomic and glycoproteomic profiles were observed between the samples.

Oncogenic KRAS expression generates a metabolic dependency on aerobic glycolysis, known as the Warburg effect. We report an effect of increased glycolytic flux that feeds into glycosphingolipid biosynthesis and is directly linked to KRAS oncogenic function. High resolution imaging and genetic approaches show that a defined subset of outer leaflet glycosphingolipids, including GM3 and SM4, is required to maintain KRAS plasma membrane localization, with GM3 engaging in cross-bilayer coupling to maintain inner leaflet phosphatidylserine content. Thus, glycolysis is critical for KRAS plasma membrane localization and nanoscale spatial organization. Reciprocally oncogenic KRAS selectively upregulates cellular content of these same glycosphingolipids, whose depletion in turn abrogates KRAS oncogenesis in pancreatic cancer models. Our findings expand the role of the Warburg effect beyond ATP generation and biomass building to high-level regulation of KRAS function. The positive feedforward loop between oncogenic KRAS signaling and glycosphingolipid synthesis represents a vulnerability with therapeutic potential.

In this work, we developed a targeted glycoproteomic method to monitor the site-specific glycoprofiles and quantities of the most abundant HDL-associated proteins using Orbitrap LC-MS for (glyco)peptide target discovery and QqQ LC-MS for quantitative analysis. We conducted a pilot study using the workflow to determine whether HDL protein glycoprofiles are altered in healthy human participants in response to dietary glycan supplementation.

Since high-density lipoprotein (HDL) glycoprofiles are associated with HDL functional capacity, we set out to determine whether diet can alter the glycoprofiles of key HDL-associated proteins, including ApoE, a potent driver of chronic disease risk. Ten healthy subjects consumed a fast food (FF) and a Mediterranean (Med) diet for 4 days in randomized order, with a 4-day wash-out between treatments. A multiple reaction monitoring method was used to characterize the site-specific glycoprofiles of HDL proteins, and HDL functional capacity was analyzed. We describe for the first time that ApoE has 7 mucin-type O-glycosylation sites, which were not affected by short-term diet. The glycoprofiles of other HDL-associated proteins were also unaffected, except that a disialylated ApoC-III glycan was enriched after Med diet, and a nonsialylated ApoC-III glycan was enriched after FF diet. Twenty-five individual glycopeptides were significantly correlated with cholesterol efflux capacity and 21 glycopeptides were correlated with immunomodulatory capacity. Results from this study indicate that the glycoprofiles of HDL-associated proteins including ApoE are correlated with HDL functional capacity but generally unaffected by diet in the short term, except ApoC-III sialylation. These results suggest that HDL protein glycoprofiles are affected by both acute and long-term factors and may be useful for biomarker discovery.