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

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

doi:10.3389/fchem.2022.839470

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.

Highlights

Glycosylation has been recognized as the most prevalent, structurally diverse, and multifunctional posttranslational modification of proteins (Moremen et al, 2012)
Previous reports suggest that stepped collision energy/higher-energy collisional dissociation (sceHCD)-MS/MS can generate the most abundant and informative fragment ions from both the peptide backbone and the attached glycan of an intact N-glycopeptide in one single spectrum compared with CID, HCD, ETD, ETciDand EThcD-MS/MS (Klein and Zaia, 2020; Riley et al, 2020)
SceHCD-MS/MS cannot provide sufficient spectral evidence for accurate location of N/O-glycosite and identification of glycan composition in the case when more than one glycosite occurs within one peptide (Zhang et al, 2021b)

Summary

INTRODUCTION

Glycosylation has been recognized as the most prevalent, structurally diverse, and multifunctional posttranslational modification of proteins (Moremen et al, 2012). Intact glycopeptide analysis can simultaneously obtain glycoprotein, glycosite, and glycan composition information It has become the major method in the glycoproteomics study. When compared with EThcD and sceHCD, EThcD-sceHCD obtained more informative fragment ions, higher spectral quality, higher Byonic score, and more intact glycopeptide identifications Does this approach work well for complex clinical samples (such as plasma, urine, cells, and tissues)? Proteins extracted from plasma from PCa patients, urine from IgAN patients, HepG2 cells, and thyroid cancer tissues were processed and digested following the filter-aided sample preparation (FASP) protocol. Analysis of variance (ANOVA) was applied to the statistical comparison among five groups in the number of intact N-glycopeptides, N-glycan compositions, and N-glycoproteins identified based on different fragmentation modes for each sample. Data were shown as means ± standard deviation (SD), and a p-value

RESULTS AND DISCUSSION

CONCLUSION

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