Glycan–protein cross-linking mass spectrometry reveals sialic acid-mediated protein networks on cell surfaces
Yixuan Xie, Siyu Chen, Qiongyu Li, Ying Sheng, Michael Russelle Alvarez, Joeriggo Reyes, Gege Xu, Kemal Solakyildirim, Carlito B. Lebrilla
Abstract
-hydroxysuccinimide (NHS)-cyclooctyne, which allowed the cross-linking of the sialic acid azides on glycans with primary amines on polypeptide backbones. The coupled peptide-glycan-peptide pairs after cross-linking were identified using the latest techniques in glycoproteomic and glycomic analyses and bioinformatics software. With this approach, information on the site of glycosylation, the glycoform, the source protein, and the target protein of the cross-linked pair were obtained. Glycoprotein-protein interactions involving unique glycoforms on the PNT2 cell surface were identified using the optimized and validated method. We built the GPX network of the PNT2 cell line and further investigated the biological roles of different glycan structures within protein complexes. Furthermore, we were able to build glycoprotein-protein complex models for previously unexplored interactions. The method will advance our future understanding of the roles of glycans in protein complexes on the cell surface.