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. 2022 Feb 24:10:839470.
doi: 10.3389/fchem.2022.839470. eCollection 2022.

Comparative N-Glycoproteomics Analysis of Clinical Samples Via Different Mass Spectrometry Dissociation Methods

Wenjuan Zeng  1 , Shanshan Zheng  1 , Tao Su  1 , Jiahan Cheng  2 , Yonghong Mao  2 , Yi Zhong  1 , Yueqiu Liu  1 , Jianhai Chen  1 , Wanjun Zhao  3 , Tianhai Lin  4 , Fang Liu  5 , Guisen Li  6 , Hao Yang  1 , Yong Zhang  1
Affiliations

Comparative N-Glycoproteomics Analysis of Clinical Samples Via Different Mass Spectrometry Dissociation Methods

Wenjuan Zeng et al. Front Chem. .

Abstract

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), higher-energy collisional dissociation-product-dependent electron-transfer dissociation (HCD-pd-ETD), and a hybrid mass spectrometry fragmentation method EThcD-sceHCD have emerged as valuable approaches for glycoprotein analysis. However, each of them incurs some compromise, necessitating the systematic performance comparisons when applied to the analysis of complex clinical samples (e.g., plasma, urine, cells, and tissues). Herein, we compared the performance of EThcD-sceHCD 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. The intact N-glycopeptides of distinct samples, namely, plasma from prostate cancer (PCa) patients, urine from immunoglobulin A nephropathy (IgAN) patients, human hepatocarcinoma cell line (HepG2), and thyroid tissues from thyroid cancer (TC) patients were analyzed by these methods. We found that EThcD-sceHCD outperformed other methods in the balance of depth and accuracy of intact N-glycopeptide identification, and sceHCD and EThcD-sceHCD have good complementarity. EThcD-sceHCD holds great potential for biomarker discovery from clinical samples.

Keywords: N-glycosylation; clinical sample; electron-transfer/higher-energy collisional dissociation (EThcD)-stepped collision energy/higher-energy collisional dissociation (sceHCD); glycoproteomics; mass spectrometry.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Schematic illustration of the workflow for human plasma, urine, cells, and tissue intact N-glycopeptides analysis using different dissociation methods.
FIGURE 2
FIGURE 2
Comparison of electron-transfer/higher-energy collisional dissociation (EThcD), stepped collision energy/higher-energy collisional dissociation (sceHCD), EThcD-sceHCD, higher-energy collisional dissociation-product-dependent electron-transfer dissociation (HCD-pd-ETD), and sceHCD-pd-ETD spectra of alpha-1-acid glycoprotein 1 N-linked glycopeptide (N93) from human plasma.
FIGURE 3
FIGURE 3
Comparison of the number of localized N-glycopeptide spectral matches (glycoPSMs), N-glycans, and intact N-glycopeptides from human plasma, urine, cells, and tissues.
FIGURE 4
FIGURE 4
Comparison of the intact N-glycopeptides identified by different tandem mass spectrometry (MS/MS) fragmentation modes.
FIGURE 5
FIGURE 5
N-glycosites (N56, N93, and N103) and deduced N-glycans were mapped in the three-dimensional structure of the alpha-1-acid glycoprotein 1 (PDB code: 3KQ0) from human plasma.
See this image and copyright information in PMC

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