Core fucosylation of N-linked glycoproteins has been linked to the functions of glycoproteins in physiological and pathological processes. However, quantitative characterization of core fucosylation remains challenging due to the complexity and heterogeneity of N-linked glycosylation. Here we report a mass spectrometry-based method that employs sequential treatment of intact glycopeptides with enzymes (STAGE) to analyze site-specific core fucosylation of glycoproteins. The STAGE method utilizes Endo F3 followed by PNGase F treatment to generate mass signatures for glycosites that are formerly modified by core fucosylated N-linked glycans. We benchmark the STAGE method and use it to characterize site specific core fucosylation of glycoproteins from human hepatocellular carcinoma and pancreatic ductal adenocarcinoma, resulting in the identification of 1130 and 782 core fucosylated glycosites, respectively. These results indicate that our STAGE method enables quantitative characterization of core fucosylation events from complex protein mixtures, which may benefit our understanding of core fucosylation functions in various diseases.

Age-related macular degeneration (AMD), Neovascular AMD (nvAMD), Nonneovascular AMD (nnvAMD), Vascular endothelial growth factor (VEGF), anti-VEGF therapy, choroidal neovascularization, treat-and-extend, pro re nata, proteomics, complement, complement factor 3 (C3), complement factor H (CFH), complement factor H-related (CHFR) proteins, apolipoprotein-B100 (ApoB100), drusen Cao et al.,2

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Most patients with neovascular age-related macular degeneration (nvAMD), the leading cause of severe vision loss in elderly US citizens, respond inadequately to current therapies targeting a single angiogenic mediator, vascular endothelial growth factor (VEGF). Here, we report that aqueous fluid levels of a second vasoactive mediator, angiopoietin-like 4 (ANGPTL4), can help predict the response of patients with nvAMD to anti-VEGF therapies. ANGPTL4 expression was higher in patients who required monthly treatment with anti-VEGF therapies compared with patients who could be effectively treated with less-frequent injections. We further demonstrate that ANGPTL4 acts synergistically with VEGF to promote the growth and leakage of choroidal neovascular (CNV) lesions in mice. Targeting ANGPTL4 expression was as effective as targeting VEGF expression for treating CNV in mice, while simultaneously targeting both was more effective than targeting either factor alone. To help translate these findings to patients, we used a soluble receptor that binds to both VEGF and ANGPTL4 and effectively inhibited the development of CNV lesions in mice. Our findings provide an assay that can help predict the response of patients with nvAMD to anti-VEGF monotherapy and suggest that therapies targeting both ANGPTL4 and VEGF will be a more effective approach for the treatment of this blinding disease.

Statistical analysis was performed with Microsoft Excel, version 16.0.16327.20248, or Prism, version 8.0, software (GraphPad). Statistical differences between 2 or multiple heterogenous groups were determined by unpaired Student's t test or 1-way or 2-way ANOVA. Analysis of data was performed using Excel.Study approval. All animal experiments were performed in accordance with the Animal Care and Use Program at Johns Hopkins University and were approved by the Institutional Animal Care and Use Committee (IACUC). All studies involving patients or patient tissue were approved by the Institutional Review Board of Johns Hopkins University School of Medicine. All participants provided written informed consent prior to enrolment in the study.

Age-related macular degeneration (AMD), Neovascular AMD (nvAMD), Vascular endothelial growth factor (VEGF), anti-VEGF therapy, choroidal neovascularization, treat-andextend, pro re nata, treatment pause Cao et al., 2