Comprehensive Plasma N-Glycoproteome Profiling Based on EThcD-sceHCD-MS/MS.
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ABSTRACT:
SUBMITTER: Mao Y
PROVIDER: S-EPMC9251008 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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.
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Project description:Protein glycosylation, one of the most heterogeneous post-translational modifications, can play a major role in cellular signal transduction and disease progression. Traditional mass spectrometry (MS)-based large-scale glycoprotein sequencing studies heavily rely on identifying enzymatically released glycans and their original peptide backbone separately, as there is no efficient fragmentation method to produce unbiased glycan and peptide product ions simultaneously in a single spectrum, and that can be conveniently applied to high throughput glycoproteome characterization, especially for N-glycopeptides, which can have much more branched glycan side chains than relatively less complex O-linked glycans. In this study, a redefined electron-transfer/higher-energy collision dissociation (EThcD) fragmentation scheme is applied to incorporate both glycan and peptide fragments in one single spectrum, enabling complete information to be gathered and great microheterogeneity details to be revealed. Fetuin was first utilized to prove the applicability with 19 glycopeptides and corresponding five glycosylation sites identified. Subsequent experiments tested its utility for human plasma N-glycoproteins. Large-scale studies explored N-glycoproteomics in rat carotid arteries over the course of restenosis progression to investigate the potential role of glycosylation. The integrated fragmentation scheme provides a powerful tool for the analysis of intact N-glycopeptides and N-glycoproteomics. We also anticipate this approach can be readily applied to large-scale O-glycoproteome characterization. Graphical Abstract ᅟ.
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