Spatial and temporal diversity of glycome expression in mammalian brain.

Mammalian brain glycome remains a relatively poorly understood area compared to other large-scale "omics" studies, such as genomics and transcriptomics due to the inherent complexity and heterogeneity of glycan structure and properties. Here, we first performed spatial and temporal analysis of glycome expression patterns in the mammalian brain using a cutting-edge experimental tool based on liquid chromatography-mass spectrometry, with the ultimate aim to yield valuable implications on molecular events regarding brain functions and development. We observed an apparent diversity in the glycome expression patterns, which is spatially well-preserved among nine different brain regions in mouse. Next, we explored whether the glycome expression pattern changes temporally during postnatal brain development by examining the prefrontal cortex (PFC) at different time point across six postnatal stages in mouse. We found that glycan expression profiles were dynamically regulated during postnatal developments. A similar result was obtained in PFC samples from humans ranging in age from 39 d to 49 y. Novel glycans unique to the brain were also identified. Interestingly, changes primarily attributed to sialylated and fucosylated glycans were extensively observed during PFC development. Finally, based on the vast heterogeneity of glycans, we constructed a core glyco-synthesis map to delineate the glycosylation pathway responsible for the glycan diversity during the PFC development. Our findings reveal high levels of diversity in a glycosylation program underlying brain region specificity and age dependency, and may lead to new studies exploring the role of glycans in spatiotemporally diverse brain functions.

Deuterium Oxide Labeling for Global Omics Relative Quantification (DOLGOReQ): Application to Glycomics.

A new relative quantification strategy for glycomics, named deuterium oxide (D2O) labeling for global omics relative quantification (DOLGOReQ), has been developed based on the partial metabolic D2O labeling, which induces a subtle change in the isotopic distribution of glycan ions. The relative abundance of unlabeled to D-labeled glycans was extracted from the overlapped isotopic envelope obtained from a mixture containing equal amounts of unlabeled and D-labeled glycans. The glycan quantification accuracy of DOLGOReQ was examined with mixtures of unlabeled and D-labeled HeLa glycans combined in varying ratios according to the number of cells present in the samples. The relative quantification of the glycans mixed in an equimolar ratio revealed that 92.4 and 97.8% of the DOLGOReQ results were within a 1.5- and 2-fold range of the predicted mixing ratio, respectively. Furthermore, the dynamic quantification range of DOLGOReQ was investigated with unlabeled and D-labeled HeLa glycans mixed in different ratios from 20:1 to 1:20. A good correlation (Pearson's r > 0.90) between the expected and measured quantification ratios over 2 orders of magnitude was observed for 87% of the quantified glycans. DOLGOReQ was also applied in the measurement of quantitative HeLa cell glycan changes that occur under normoxic and hypoxic conditions. Given that metabolic D2O labeling can incorporate D into all types of glycans, DOLGOReQ has the potential as a universal quantification platform for large-scale comparative glycomic experiments.

Comprehensive Profiling of Surface Gangliosides Extracted from Various Cell Lines by LC-MS/MS.

Gangliosides act as a surface marker at the outer cellular membrane and play key roles in cancer cell invasion and metastasis. Despite the biological importance of gangliosides, they have been still poorly characterized due to the lack of effective analytical tools. Herein, we performed molecular profiling and structural elucidation of intact gangliosides in various cell lines including CFPAC1, A549, NCI-H358, MCF7, and Caski. We identified and quantified a total of 76 gangliosides on cell membrane using C18 LC-MS/MS. Gangliosides found in each cell line exhibited high complexity and diversity both qualitatively and quantitatively. The most abundant species was GM3(d34:1) in CFPAC1, NCI-H358, and MCF7, while GM2(d34:1) and GM1(d34:1) were major components in A549 and Caski, respectively. Notably, glycan moieties showed more diversity between cancer cell lines than ceramide moieties. In addition, noncancerous pancreatic cell line (hTERT/HPNE) could be distinguished by gangliosides containing different levels of sialic acid compared with cancerous pancreatic cell line (CFPAC1). These results clearly demonstrated the feasibility of our analytical platform to comprehensive profile of cell surface gangliosides for identifying cell types and subgrouping cancer cell types.