Characterization of the glycosylation pattern of HAVCR2 and OSTP following a glycoproteomics approach

Protein glycosylation is a common post-translational modification, involving glycan linkage to proteins via N- or O- linkages. It affects protein folding and stability, trafficking, differentiation, and cell–cell interactions, among others. In addition, aberrant protein glycosylation is a hallmark of cancer, crucial in malignant transformation, tumor development, invasiveness, and metastatic disease. The characterization of the glycosylation pattern of proteins directly involved in immune response-related processes, such as recognition of pathogens or tumoral cells, might provide valuable information for the understanding of this interaction process, helping drug or vaccine design.

In this regard, MS-based approaches generate complementary information to NMR-based studies, providing specific site-assigment and occupancy information. In this work we present data on the glycan profile characterization of two biologically and clinically relevant overexpressed and purified proteins (HAVCR2 and OSTP), with important roles in immune response modulation and cell-cell interaction. Trypsin and chymotrypsin were used for their digestion, and the resulting peptides were analysed in a timsTOF Pro mass spectrometer (Bruker). Specific acquisition methods were used for the analysis of unmodified peptides and for the glycopeptides. The data was analysed using Byonic software (Protein Metrics).

Altogether, our results reveal that the use of different proteases, together with specific MS acquisition methods, and efforts on the bioinformatic side (scan merging, wildcard search for unexpected glycan modifications or variants in Byonic) provide deep information on the glycosylation pattern of HAVCR2 and OSTP, that complements and details information obtained by NMR.