EThcD-sceHCD-MS/MS-based Clinical N-glycoproteomics

Site-specific N-glycosylation characterization requires intact N-glycopeptide analysis based on suitable tandem mass spectrometry (MS/MS) method. Electron-transfer/higher-energy collisional dissociation (EThcD), stepped collision energy/higher-energy collisional dissociation (sceHCD), and higher-energy collision dissociation-product-dependent electron-transfer dissociation (HCD-pd-ETD) have emerged as valuable approaches for clinical glycoproteomics. However, each of them incurs some compromise, necessitating the performance comparisons when applied to the analysis of complex clinical samples (e.g., plasma, urine, cells, and tissue). Herein, we developed a hybrid mass spectrometry fragmentation method, EThcD-sceHCD, by combining EThcD and sceHCD. Moreover, we compared the performance of this method with those previous approaches (EThcD, sceHCD, HCD-pd-ETD, and sceHCD-pd-ETD) in the intact N-glycopeptide analysis, and determined its applicability for clinical N-glycoproteomic study.