Comprehensive human plasma N-glycoproteome profiling based on EThcD-sceHCD-MS/MS

N-glycoproteins are involved in various biological processes. More than one-third of plasma tumor protein biomarkers approved by the Food and Drug Administration (FDA) are glycoproteins which would enhance the specificity and/or sensitivity of cancer diagnosis. Therefore, the characterization of plasma N-glycoproteome is essential. In previous studies, we developed an integrated method based on combinatorial peptide ligands library (CPLL) and stepped collision energy/higher-energy collisional dissociation (sceHCD) for in-depth and comprehensive N‑glycoproteome profiling of human plasma. Recently, we presented a new mass spectrometry fragmentation method (EThcD-sceHCD) which outperformed sceHCD in the accuracy of identification. Herein, we integrated CPLL into EThcD-sceHCD, and compared the performance of EThcD-sceHCD with those previous approaches (EThcD and sceHCD) in pooled prostatic cancer (PCa) patients’ plasma intact N-glycopeptide analysis. We found that EThcD-sceHCD outperformed other methods in both the depth and accuracy of intact N-glycopeptides identification, and sceHCD and EThcD-sceHCD have good complementarity. Combining sceHCD and EThcD-sceHCD can cover almost all glycoproteins and intact N-glycopeptides. Our study holds great potential for human plasma biomarker discovery.