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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.

Gastric cancer (GC) is one of the leading causes of cancer-related death worldwide, largely because of difficulties in early diagnosis. Despite accumulating evidence indicating that aberrant glycosylation is associated with GC, site-specific localization of the glycosylation to increase specificity and sensitivity for clinical use is still an analytical challenge. Here, we created an analytical platform with a targeted glycoproteomic approach for GC biomarker discovery. Unlike the conventional glycomic approach with untargeted mass spectrometric profiling of released glycan, our platform is characterized by three key features: it is a target-protein-specific, glycosylation-site-specific, and structure-specific platform with a one-shot enzyme reaction. Serum haptoglobin enriched by immunoaffinity chromatography was subjected to multispecific proteolysis to generate site-specific glycopeptides and to investigate the macroheterogeneity and microheterogeneity. Glycopeptides were identified and quantified by nano liquid chromatography-mass spectrometry and nano liquid chromatography-tandem mass spectrometry. Ninety-six glycopeptides, each corresponding to a unique glycan/glycosite pairing, were tracked across all cancer and control samples. Differences in abundance between the two groups were marked by particularly high magnitudes. Three glycopeptides exhibited exceptionally high control-to-cancer fold changes along with receiver operating characteristic curve areas of 1.0, indicating perfect discrimination between the two groups. From the results taken together, our platform, which provides biological information as well as high sensitivity and reproducibility, may be useful for GC biomarker discovery. Graphical abstract ᅟ.