Post-translational modifications (PTMs) alter the functional properties of proteins and thus regulate important cellular processes. One of the more difficult PTMs to investigate also happens to be one of the most common: glycosylation derived from the endoplasmic reticulum and Golgi apparatus. Although characterizing this type of PTM poses technical challenges, the clinical interest in protein pharmaceuticals and the potential for the discovery of novel biomarkers render it a salient area of study.
Recently, Trinidad et al. (2013) conducted the first exhaustive characterization of N- and O-glycosylation using in vivo murine samples to identify sites and glycans for hundreds of proteins from the synaptosome.1 Previously, Trinidad et al. had created a workflow for the simultaneous analysis of two PTMs that play signaling and regulatory roles, phosphorylation and GlcNAcylation. Given that the synaptosome contains membrane-bound proteins, the researchers now turned this enrichment method to glycosylation and demonstrated that lectin-affinity fractionation using wheat germ agglutinin indeed enriches N- and O-linked glycans acquired from the endoplasmic reticulum and Golgi apparatus. After enrichment, the samples were subjected to liquid chromatography–mass spectrometry with electron transfer dissociation (Thermo Scientific).
Ideally, researchers prefer to fully characterize a glycopeptide by revealing the specific sequence of the peptide, the site where the modification occurs, and the precise glycan attached at the site. However, current methods make this level of analysis difficult. Collision-induced dissociation, for instance, cleaves glycosidic bonds and requires researchers to contend with spectra littered with carbohydrate fragments that must be interpreted manually. Even electron transfer dissociation, as used in this study, is not without its drawbacks, as it generally requires researchers to enumerate specific mass values for potential glycans prior to analysis.
Trinidad et al. offer a novel, iterative approach to data analysis that allows researchers to use the obtained spectral data to identify the most likely glycans while stopping short of precisely characterizing specific glycans and bonds. Their method uses a series of unknown modification searches to filter data for likely modifications and match those mass values to common carbohydrate structures with implied linkages.
Overall, the researchers were able to determine both N- and O-glycosylation and a handful of relatively rare PTMs such as O-mannosylation, EGF domain-specific O-fucosylation, and tyrosine O-glycosylation. Indeed, Trinidad et al. used their method for data interpretation to identify over 2,500 unique N- and O-linked glycopeptides on 453 proteins. The data provide insight into the importance of glycosylation to the function of the synaptosome and the existence of micro-heterogeneity (with up to 19 unique oligosaccharides identified at each site) as a site-specific feature.
1. Trinidad, J., et al. (2013) “N- and O- glycosylation in the murine synaptosome,” Molecular and Cellular Proteomics, in press July 1 as Manuscript M113.030007.
Post Author: Melissa J. Mayer. Melissa is a freelance writer who specializes in science journalism. She possesses passion for and experience in the fields of proteomics, cellular/molecular biology, microbiology, biochemistry, and immunology. Melissa is also bilingual (Spanish) and holds a teaching certificate with a biology endorsement.