N-linked glycosylation is a ubiquitous protein modification that is capable of modulating protein structure, function and interactions. Many proteins in the brain associated with the synapse and important for synaptic transmission are highly glycosylated and their glycosylation could be important for learning and memory related molecular processes and synaptic plasticity. In the present study, we extend the knowledge of the synaptic glycome and glycoproteome by performing glycan- and intact glycopeptide-focused analyses of isolated rat nerve terminals (synaptosomes) by LC-MS/MS. Overall, glycomics identified a total of 41N-glycans in isolated synaptosomes. SialylatedN-glycans represented only 7% of the total abundance of the rat synaptosomeN-glycome with oligomannose, neutral hybrid and complex typeN-glycans being the most abundant structures. Using detergent extraction of the active zone proteins from the synaptosomes revealed a change in the active zone glycan abundance in comparison with the rest of the synaptosome glycan content. Characterization of intact sialylatedN-linked glycopeptides enriched by titanium dioxide chromatography revealed more than 85% selectivity of sialylated species and the presence of NeuGc on active zone proteins. In addition, both disialic and trisialic acid modified glycans were present on synaptic glycoproteins, although oxonium ion profiling revealed that trisialic units were only present on glycoproteins in the detergent soluble fraction. However, correct identification of intact sialylatedN-linked glycopeptides using the Byonic program failed, most likely due to the lack of peptide backbone fragmentation during tandem mass spectrometry.
|Status||Udgivet - aug. 2021|
Bibliografisk noteFunding Information:
This study was supported by the Danish Natural Science Research Council (grant number: 6108-00621B). The Villum Center for Bioanalytical Sciences at SDU is acknowledged for access to state-of-the art mass spectrometric instrumentation. Furthermore, the collaboration between the University of Southern Denmark and Griffith University was supported by a Boehringer Ingelheim Fonds travel grant. DK is the recipient of an Australian Research Council Future Fellowship (project number FT160100344) funded by the Australian Government. TO is the recipient of a Griffith University Postgraduate Research Scholarship (GUPRS).
© The Royal Society of Chemistry 2021.