Comprehensive Plasma N-Glycoproteome Profiling Based on EThcD-sceHCD-MS/MS

Glycoproteins are involved in a variety of biological processes. More than one-third of the plasma protein biomarkers of tumors approved by the FDA are glycoproteins, and could improve the diagnostic specificity and/or sensitivity. Therefore, it is of great significance to perform the systematic characterization of plasma N-glycoproteome. In previous studies, we developed an integrated method based on the combinatorial peptide ligand library (CPLL) and stepped collision energy/higher energy collisional dissociation (sceHCD) for comprehensive plasma N-glycoproteome profiling. Recently, we presented a new fragmentation method, EThcD-sceHCD, which outperformed sceHCD in the accuracy of identification. Herein, we integrated the combinatorial peptide ligand library (CPLL) into EThcD-sceHCD and compared the performance of different mass spectrometry dissociation methods (EThcD-sceHCD, EThcD, and sceHCD) in the intact N-glycopeptide analysis of prostate cancer plasma. The results illustrated that EThcD-sceHCD was better than EThcD and sceHCD in the number of identified intact N-glycopeptides (two-folds). A combination of sceHCD and EThcD-sceHCD methods can cover almost all glycoproteins (96.4%) and intact N-glycopeptides (93.6%), indicating good complementarity between the two. Our study has great potential for medium- and low-abundance plasma glycoprotein biomarker discovery.