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Microwave-assisted deglycosylation for rapid and sensitive analysis of N-glycans via glycosylamine derivatization

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Abstract

A rapid and sensitive N-glycan profiling strategy for MALDI-MS incorporating the use of deglycosylation with microwave assistance and the co-derivatization of glycosylamine labeling with tris(2,4,6-trimethoxyphenyl)phosphonium acetic acid N-hydroxysuccinimide ester (TMPP-Ac-OSu) and methylamidation has been developed in this work. Notably, highly efficient release and tagging of N-glycans from ribonuclease B was achieved in less than 90 min, providing up to 35-fold enhancement of MALDI-MS sensitivity with comparison to underivatized N-glycans. After further validation with other two standard glycoproteins (ovalbumin and bovine fetuin), the proposed strategy was applied to human serum for preliminary pathological analysis of N-glycans between healthy and lung cancer individuals. As a result, significant differences (T test p value <0.01) of 6 glycan structures were determined from 54 detected N-glycan structures with only 50 nL of loading amount and further confirmed through PCA and ROC (AUC) analyses between two sample sets. Subsequently, the trend of each lung cancer stage and controls in expression of the selected glycans was implemented with T test and box-plots. Accordingly, these structures can be used as potential lung cancer glycan-based biomarkers and for further definition of cancer progression highlighting the ability of proposed method to rapidly and efficiently analyze N-glycome present in human serum.

MALDI-TOF MS analysis of N-glycans by microwave-assisted deglycosylation and glycosylamine derivatization

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Abbreviations

ACN:

Acetonitrile

AUC:

Area under the ROC curve; T test Student’s t test

DHB:

2,5-Dihydroxybenzoic acid

DMSO:

Dimethyl sulfoxide

MALDI-MS:

Matrix-assisted laser desorption/ionization mass spectrometry

MCC:

Microcrystalline cellulose

NP-HPLC:

Normal phase high-performance liquid chromatography

NP-40:

Octylphenoxypolyethoxyethanol

PCA:

Principal component analysis

PNGase F:

Peptide-N-glycosidase F

PyAOP:

(7-Azabenzotriazol-1-yloxy) trispyrrolidinophosphonium hexafluorophosphate

RNase B:

Ribonuclease B

ROC:

Receiver operating characteristic curve

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

TFA:

Trifluoroacetic acid

TMPP-Ac-OSu:

Tris(2,4,6-trimethoxyphenyl)phosphonium acetic acid N-hydroxysuccinimide ester

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Acknowledgements

The authors gratefully acknowledge the financial supports from the National Natural Science Foundation of China (20905027 and 81402198).

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Author notes

  1. Yike Wu and Chang Wang contributed equally to this work.

Authors and Affiliations

  1. Britton Chance Center for Biomedical Photonics at Wuhan National Laboratory for Optoelectronics—Hubei Bioinformatics & Molecular Imaging Key Laboratory, Systems Biology Theme, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, 1037 Luoyu Road, Wuhan, Hubei, 430074, China

    Yike Wu, Chang Wang, Yanyan Liu, Liang Zhang, Yun Xia, Xiaojun Feng, Bi-Feng Liu & Xin Liu

  2. Laboratory of Hubei International Travel Healthcare Center, Wuhan, Hubei, 430070, China

    Jing Luo

  3. School of Chemistry, Chemical Engineering and Life Sciences, Wuhan University of Technology, Wuhan, Hubei, 430070, China

    Yawei Lin

Authors
  1. Yike Wu
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  2. Chang Wang
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  3. Jing Luo
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  4. Yanyan Liu
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  5. Liang Zhang
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  8. Bi-Feng Liu
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  9. Yawei Lin
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  10. Xin Liu
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Corresponding authors

Correspondence to Yawei Lin or Xin Liu.

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The study was carried out in accordance with the Helsinki Declaration and informed consents were obtained from the participants in accordance with the study protocols approved by the Ethics Committee of Huazhong University of Science and Technology.

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The authors declare that they have no conflicts of interest.

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Wu, Y., Wang, C., Luo, J. et al. Microwave-assisted deglycosylation for rapid and sensitive analysis of N-glycans via glycosylamine derivatization. Anal Bioanal Chem 409, 4027–4036 (2017). https://doi.org/10.1007/s00216-017-0346-2

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  • DOI: https://doi.org/10.1007/s00216-017-0346-2

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