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

Abstract

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.

Keywords: EThcD-sceHCD; N-glycoproteomics; combinatorial peptide ligand library; mass spectrometry; plasma.

FIGURE 1

Schematic representation of the workflow for human plasma LAP intact N-glycopeptide analysis using different dissociation methods (EThcD, sceHCD, and EThcD-sceHCD).

FIGURE 2

Comparison of the number of N-glycoPSMs, N-glycans, intact N-glycopeptides, and N-glycoproteins from pooled PCa patients’ plasma using different dissociation methods. (ANOVA was used for the statistical comparison among three groups, and Student’s t-test was used for the statistical comparison between two groups. The homogeneity test was performed. The error bar denotes SD.)

FIGURE 3

Comparison of the EThcD (A), secHCD (B), and EThcD-sceHCD (C) spectra of prothrombin intact N-glycopeptides (N143) from human plasma.

FIGURE 4

Global analysis of identified plasma N-glycoproteins. (A) Heat map of identified plasma N-glycoproteins using different fragmentation modes. Colored lines indicate the number of repetitions identified. (B) Gene ontology (GO) biological process (BP), cellular component (CC), and molecular function (MF) enrichment analysis.

FIGURE 5

Global analysis of identified plasma intact N-glycopeptides. (A) Comparison of identified intact N-glycopeptides using EThcD, secHCD, and EThcD-sceHCD. (B) N-glycosites (N121, N143, and N416) and deduced N-glycans were demonstrated in the three-dimensional structure of the prothrombin (PDB code: 1A2C) from pooled PCa patients’ plasma.