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Highly efficient N-glycoproteomic sample preparation by combining C18 and graphitized carbon adsorbents

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Abstract

Conventional N-glycoproteome analysis usually applies C18 reversed-phase (RP) adsorbent for sample purification, which will lead to unavoidable sample loss due to the high hydrophilicity of N-glycopeptides. In this study, a porous graphitized carbon (PGC) absorbent was combined with a C18 adsorbent for N-glycopeptide purification in comprehensive N-glycoproteome analysis based on the hydrophobic and polar interactions between carbon and N-glycans. It was observed that the small hydrophilic N-glycopeptides that cannot retain onto C18 adsorbent can be captured by the graphitized carbon, while the large hydrophobic N-glycopeptides that cannot retain onto the graphitized carbon can be feasibly captured by the C18 adsorbent. Comparing with sample purification by using C18 adsorbent only, 28.5 % more N-glycopeptides were identified by combining both C18 and PGC adsorbents. The C18-PGC strategy was further applied for both sample purification and pre-fractionation of a complex protein sample from HeLa cell. After hydrophilic interaction chromatography enrichment, 1,484 unique N-glycopeptides with 1,759 unique N-glycosylation sites were finally identified.

The overlap of identified N-glycosylation sites by different SPE strategies

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Abbreviations

ACN:

Acetonitrile

CID:

Collision-induced dissociation

Click maltose-HILIC:

Click maltose Hydrophilic interaction chromatography

DTT:

Dithiothreitol

FA:

Formic acid

HILIC:

Hydrophilic interaction chromatography

HRP:

Horseradish peroxidase

IAA:

Iodoacetamide

LC-MS:

Liquid chromatography coupled with mass spectrometry

LTQ:

Linear ion trap

MALDI-TOF MS:

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry

PGC:

Porous graphitized carbon

PNGase F:

Peptide N-glycosidase F

RP:

Reversed phase

SPE:

Solid-phase extraction

TFA:

Trifluoroacetic acid

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Acknowledgments

The authors greatly appreciate Dr. Xinmiao Liang and Dr. Zhimou Guo for providing the HILIC materials as a gift. Financial support is gratefully acknowledged from the China State Key Basic Research Program Grant (2013CB911203 and 2012CB910601), the financial support from the NSFC (21021004, 21105101 and 21235006), the Analytical Method Innovation Program of MOST (2012IM030900) to H. Zou, and the financial support from NSFC (21305139) and “Hundred Talent Young Scientist Program” by DICP to F. Wang.

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Authors and Affiliations

  1. Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), 116023, Dalian, China

    Jing Liu, Fangjun Wang, Jun Zhu, Jiawei Mao, Zheyi Liu, Kai Cheng, Hongqiang Qin & Hanfa Zou

  2. University of Chinese Academy of Sciences, 100049, Beijing, China

    Jing Liu, Jun Zhu, Jiawei Mao, Zheyi Liu & Kai Cheng

Authors
  1. Jing Liu
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  2. Fangjun Wang
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  3. Jun Zhu
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  4. Jiawei Mao
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  5. Zheyi Liu
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  6. Kai Cheng
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  7. Hongqiang Qin
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  8. Hanfa Zou
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Correspondence to Fangjun Wang or Hanfa Zou.

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Liu, J., Wang, F., Zhu, J. et al. Highly efficient N-glycoproteomic sample preparation by combining C18 and graphitized carbon adsorbents. Anal Bioanal Chem 406, 3103–3109 (2014). https://doi.org/10.1007/s00216-014-7716-9

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  • DOI: https://doi.org/10.1007/s00216-014-7716-9

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