(736f) Synglycan: A Glycomics Reporter Toolkit for Multiple Cell Types and In Vivo application

Glycosylation is a ubiquitous post-translational modification in nature. The structural characterization of glycans is complicated due to the branched, non-linear structure of the carbohydrates, and their heterogeneous expression among metazoans. In order to simplify the non-destructive analysis of glycans, we developed an experimental toolkit called SynGlycan, along with a software application for mass spectrometry based data analysis of site-specific glycosylation. The SynGlycan toolkit includes a mixture of naturally occurring N- and O-linked glycopeptides from cancer related entities (MUC-16, MUC-1, PSA/prostate-specific antigen), common glycoproteins (RNAse B, CD43), HIV Gp120 and human IgG1. When introduced into heterologous cells as Fc-fusion proteins, the Golgi processed SynGlycan probes are secreted and can be purified using a straightforward scheme. The final product can then be analyzed using high resolution, high sensitivity, quantitative mass spectrometry (MS) with the Orbitrap Fusion Lumos mass analyzer. The implemented MS workflow includes high resolution (<10ppm) precursor mass measurement that is followed by beam-type collision induced dissociation (i.e. HCD). If glycan oxonium ions are produced, further MS/MS fragmentation analysis is performed using both standard collision induced dissociation (CID) and Electron-Transfer/High-Energy Collision Dissociation (EThcD). Data obtained are analyzed using a variant of the GlycoProteomics Analysis Toolbox (ref. 1) that records glycopeptide retention time and label free quantitative data. A comparison of the SynGlycan glycoproteomics profile obtained from HEK293 cells with parallel N-glycomics data obtained from the same cells following PNGaseF release, demonstrates that the SynGlycan probe semi-quantitatively captures 53 of the 73 glycans present on the cells. Lentiviral expression of the SynGlycan probes in six breast cancer cell lines enabled straightforward analysis of the cellular glycan profile and metabolic comparisons that could be related to disease molecular phenotype. In a final example, that SynGlycan reagents were applied in vivo in mouse tumor models to capture the evolution of carbohydrate structures as breast tumors grow and metastasize. Overall, the SynGlycan toolkit provides a streamlined, novel molecular technology to longitudinally assay cellular glycosylation and metabolic pathways, with focus on dynamic glycan structure change ex vivo and in vivo. The simplicity of the approach and the ready availability of software resource could enable its broad usage in biomedical research.

Reference:

(1) Liu et al. “A comprehensive, open-source platform for mass spectrometry based glycoproteomics data analysis”, Mol. Cell. Proteomics, 16(11): 2037-47, 2017.