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.

Aberrant glycosylation has been extensively reported in cancer, with fundamental changes in the glycosylation patterns of cell-surface and secreted proteins largely occurring during cancer progression. As such, serum glycan and glycopeptide biomarkers have been discovered using mass spectrometry and proposed for cancer detection. Here, we report for the first time potential serum N-glycan and glycopeptide biomarkers for Philippine lung cancer patients. The N-glycan and glycoprotein profiles of a cohort (n = 26 patients, n = 22 age- and gender-matched) of lung cancer patients were analyzed and compared to identify potential N-glycan and glycopeptide serum biomarkers using nano-QToF-MS/MS and ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry dynamic multiple monitoring methods, respectively. Statistical analyses identified differential N-glycan and glycopeptide abundances. The N-glycans were mostly sialylated and sialofucosylated branched structures. The glycopeptides involved proteins in complement and coagulation cascades (p _adj = 6.418 × 10^-4), innate immunity (p _adj = 6.094 × 10^-3), acute inflammatory response (p _adj = 6.404 × 10^-5), defense response (p _adj = 2.082 × 10^-4), complement activation pathways (p _adj = 1.895 × 10^-2), and immunoglobulin-mediated immune response pathways (p _adj = 4.818 × 10^-2). Biomarker models were constructed using serum N-glycans [area under the curve (AUC) = 0.775; 95% CI: 0.617-0.931] and glycopeptides (AUC = 0.959; 95% CI: 0.85-1.0), with glycopeptides having higher accuracies than N-glycans. The results suggest that in the Philippine lung cancer patient sera, specific N-glycans and site-specific glycans are differentially expressed between cases and controls. This report represents the first serum glycan and glycopeptide biomarkers of Philippine lung cancer patients, further demonstrating the utility of mass spectrometry-based glycomic and glycoproteomic methods.

Glycoproteins are an underexploited drug target for cancer therapeutics. In this work, we integrated computational methods in network pharmacology and in silico docking approaches to identify phytochemical compounds that could potentially interact with several cancer-associated glycoproteins. We first created a database of phytochemicals from selected plant species, Manilkara zapota (sapodilla/chico), Mangifera indica (mango), Annona muricata (soursop/guyabano), Artocarpus heterophyllus (jackfruit/langka), Lansium domesticum (langsat/lanzones), and Antidesma bunius (bignay), and performed pharmacokinetic analysis to determine their drug-likeness properties. We then constructed a phytochemical-glycoprotein interaction network and characterized the degree of interactions between the phytochemical compounds and with cancer-associated glycoproteins and other glycosylation-related proteins. We found a high degree of interactions from α-pinene (Mangifera indica), cyanomaclurin (Artocarpus heterophyllus), genistein (Annona muricata), kaempferol (Annona muricata and Antidesma bunius), norartocarpetin (Artocarpus heterophyllus), quercetin (Annona muricata, Antidesma bunius, Manilkara zapota, Mangifera indica), rutin (Annona muricata, Antidesma bunius, Lansium domesticum), and ellagic acid (Antidesma bunius and Mangifera indica). Subsequent docking analysis confirmed that these compounds could potentially bind to EGFR, AKT1, KDR, MMP2, MMP9, ERBB2, IGF1R, MTOR, and HRAS proteins, which are known cancer biomarkers. In vitro cytotoxicity assays of the plant extracts showed that the n-hexane, ethyl acetate, and methanol leaf extracts from A. muricata, L. domesticum and M. indica gave the highest growth inhibitory activity against A549 lung cancer cells. These may help further explain the reported cytotoxic activities of select compounds from these plant species.

Cancer progression is linked to aberrant protein glycosylation due to the overexpression of several glycosylation enzymes. These enzymes are underexploited as potential anticancer drug targets and the development of rapid-screening methods and identification of glycosylation inhibitors are highly sought. An integrated bioinformatics and mass spectrometry-based glycomics-driven glycoproteomics analysis pipeline was performed to identify an N-glycan inhibitor against lung cancer cells. Combined network pharmacology and in silico screening approaches were used to identify a potential inhibitor, pictilisib, against several glycosylation-related proteins, such as Alpha1-6FucT, GlcNAcT-V, and Alpha2,6-ST-I. A glycomics assay of lung cancer cells treated with pictilisib showed a significant reduction in the fucosylation and sialylation of N-glycans, with an increase in high mannose-type glycans. Proteomics analysis and in vitro assays also showed significant upregulation of the proteins involved in apoptosis and cell adhesion, and the downregulation of proteins involved in cell cycle regulation, mRNA processing, and protein translation. Site-specific glycoproteomics analysis further showed that glycoproteins with reduced fucosylation and sialylation were involved in apoptosis, cell adhesion, DNA damage repair, and chemical response processes. To determine how the alterations in N-glycosylation impact glycoprotein dynamics, modeling of changes in glycan interactions of the ITGA5–ITGB1 (Integrin alpha 5-Integrin beta-1) complex revealed specific glycosites at the interface of these proteins that, when highly fucosylated and sialylated, such as in untreated A549 cells, form greater hydrogen bonding interactions compared to the high mannose-types in pictilisib-treated A549 cells. This study highlights the use of mass spectrometry to identify a potential glycosylation inhibitor and assessment of its impact on cell surface glycoprotein abundance and protein–protein interaction.

Sphingolipids are an essential subset of bioactive lipids found in most eukaryotic cells that contribute to membrane biophysical properties and are involved in cellular differentiation, recognition, and mediating interactions. The described nanoHPLC-ESI-Q/ToF methodology utilizes known biosynthetic pathways, accurate mass detection, optimized collision-induced disassociation, and a robust nanoflow chromatographic separation for the analysis of intact sphingolipids found in human tissue, cells, and serum. The methodology was developed and validated with an emphasis on addressing the common issues experienced in profiling these amphipathic lipids, which are part of the glycocalyx and lipidome. The high sensitivity obtained using nanorange flow rates with robust chromatographic reproducibility over a wide range of concentrations and injection volumes results in confident identifications for profiling these low-abundant biomolecules.