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Highlights

• High throughput glycoproteomics method with multiplexed quantification

• 25-fold improvement of the mouse brain glycoproteome coverage

• Structural features dictate level of glycosite micro-heterogeneity

• Gut microbiome composition extensively impacts the brain glycoproteome

• Modulation of glycosylation is site-specific

Protein glycosylation is a highly diverse post-translational modification, modulating key cellular processes such as cell signaling, adhesion and cell-cell interactions. Its deregulation has been associated with various pathologies, including cancer and neurological diseases. Methods capable of quantifying glycosylation dynamics are essential to start unraveling the biological functions of protein glycosylation. Here we present Deep Quantitative Glycoprofiling (DQGlyco), a method that combines high-throughput sample preparation, high-sensitivity detection, and precise multiplexed quantification of protein glycosylation. We used DQGlyco to profile the mouse brain glycoproteome, in which we identify 158,972 and 15,056 unique N- and O-glycopeptides localized on 3,199 and 2,365 glycoproteins, respectively - this amounts to 25-fold more glycopeptides identified compared to previous studies. We observed extensive heterogeneity of glycoforms and determined their functional and structural preferences. The presence of a defined gut microbiota resulted in extensive remodeling of the brain glycoproteome when compared to that of germ-free animals, exemplifying how the gut microbiome may affect brain protein functions.