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GlycoproteomeDB

Mouse Brain Spatial Glycoproteome Atlas

Hippocampus Olfactory Bulb Rest Brain Rest Brain Rest Brain Rest Brain Rest Brain Rest Brain Hypothalamus Hypothalamus Thalamus Hypophysis Midbrain Midbrain Cerebellum Cerebellum Pons Pons Pons Medulla Choroid Plexus Cerebral Cortex Cerebral Cortex

Glycoproteomics analysis is essential for understanding the roles of glycoproteins in biological systems, yet current methods demand high sample amounts and extensive glycopeptide enrichment, limiting their application in biological studies and high-throughput analysis. This paper introduces a GlycoDIA method, which allows for rapid, sensitive, and reproducible O-glycoproteomic analysis. Here we specifically focused on brain tissue analysis addressing the need to reduce sample requirements, enhance high-throughput capabilities, and improve glycopeptide identification accuracy. We developed a comprehensive spectral library using HCD fragmentation and implemented a precursor subtraction method within the DIA pipeline, presenting glycopeptides as non-glycosylated peptides with cumulative glyco compositions. Applying this strategy, we analyzed ten mouse brain compartments from three biological replicates, producing a quantitative atlas of mouse brain compartments.

This atlas is presented as an interactive web-based database for querying glycoprotein candidates, representing a significant advancement in glycoproteomics and its application to brain tissue analysis.

The Web Atlas consists of three interactive subpages, allowing the user to explore general information on the compartments analysed, compare compartements pairwise or against the entire brain and provides visualizations of the expression profiles of glycopeptides and proteomes on all genes.

Compartment Info provides a summary of each compartment and its 10 highest expressed glycopeptides and proteomes displayed in a bar chart. The user can select compartments of interest to display the respective information and hover over the bar chart to access more detailed information on the values.

Compare View allows the user to compare compartments pairwise or against the entire brain. For this, the user either clicks on two compartments of interes or one compartment and activates the Entire Brain switch. The data is visualiezed in a volcano plot, indicating down- and up regulated genes with red and green color, respectively. The regulated genes with precursor ID and their dolf change are shown in two sorted lists below the graphics. With two switch buttons, the user can set the p-value (0.05 or 0.01) and the fold change (2 or 4, or 1 and 2 on the log2 scale) to their desired setting. Selecting an entry from the down- and up-regulated tabels, redirects the user to the corresponding entry in the Expression Map.

Expression Map vizualieses the brain expression values in all measured compartmens. The user enters a gene in the input field at the top of the page and confirms their choise with the button. The two brains on the left visualie the expression values of glycopeptides and proteomes using a colored scale that is indicated next to the brains. A bar chart of these values allows for better comparison of the values and also shows error bars. Hovering over the brains compartmens or the bars in the chart will allow the user to access the numerical values of the expressions. In the table the user can browse through all available sequences for that gene and their glyco composition. The position of each sequence will be displayed in that table as well as in the sequence visualization below. Selecting a sequence and a glyco composition from the table allows the user to display the respective information in the brains and chart. The sequence visualization below indicates the position of the selected sequence in the gene and also provides redirection to the respective AlphaFold structure by clicking on the link next to the sequence header.