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In-depth analysis of site-specific N-glycosylation in vitronectin from human plasma by tandem mass spectrometry with immunoprecipitation

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Abstract

The characterization of site-specific microheterogeneity in glycoprotein is very important for understanding cell biology and disease processes. Vitronectin is well known to be a multifunctional glycoprotein in the blood and the extracellular matrix, which is related to hepatocellular carcinoma (HCC). Here, we systematically analyzed the site-specific N-glycopeptides of vitronectin in human plasma by tandem mass spectrometry combined with immunoprecipitation and hydrophilic interaction liquid chromatography (HILIC) enrichment. Vitronectin was purified with immunoprecipitation by monoclonal antibody from plasma and digested to tryptic N-glycopeptides.Then, enrichment with HILIC materials was used and followed by analysis with nano-LC/MS/MS. The sequences of N-glycopeptides were identified from the mass spectra by high-energy C-trap dissociation (HCD) and collision-induced dissociation (CID). In HCD mode, oxonium ions were used for recognizing glycopeptides and y ions for sequencing the peptide backbone. In CID mode, Y ions were used for characterizing their glycoforms. As a result, a total of 17 site-specific N-glycopeptides were completely identified in all of the three N-glycosylation sites of vitronectin in human plasma, including 12 N-glycopeptides first reported. Finally, we specifically found that three hybrid and four complex glycopeptides of triantennary forms with outer fucosylation increased in HCC human plasma.

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Abbreviations

CID:

Collision-induced dissociation

Fuc:

Fucose

Gal:

Galactose

GlcNAc:

N-acetylglycosamine

HCC:

Hepatocellular carcinoma

HCD:

High-energy C-trap dissociation

HILIC:

Hydrophilic interaction liquid chromatography

IP:

Immunoprecipitation

Man:

Mannose

N:

Asparagine

Neu5Ac:

N-acetyl neuraminic acid

NGSL~:

NGSLFAFR

NISD~:

NISDGFDGIPDNVDAALALPAHSYSGR

NNAT~:

NNATVHEQVGGPSLTSDLQAQSK

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Acknowledgments

The research was supported by the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI13C2098); the research program through the Korea Basic Science Institute (grant number: D34413, T34750); and the Proteogenomic Research Program, through the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2013M3A9B9044431).

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Authors and Affiliations

  1. Mass Spectrometry Research Center, Korea Basic Science Institute, Chungbuk, Republic of Korea

    Heeyoun Hwang, Ju Yeon Lee, Hyun Kyoung Lee, Gun Wook Park, Hoi Keun Jeong, Jin Young Kim & Jong Shin Yoo

  2. Department of Chemistry, Yonsei University, Seoul, Republic of Korea

    Ju Yeon Lee & Myeong Hee Moon

  3. Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, Republic of Korea

    Hyun Kyoung Lee, Gun Wook Park, Hoi Keun Jeong & Jong Shin Yoo

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  1. Heeyoun Hwang
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  2. Ju Yeon Lee
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Correspondence to Jin Young Kim or Jong Shin Yoo.

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Heeyoun Hwang and Ju Yeon Lee contributed equally to this work.

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Hwang, H., Lee, J.Y., Lee, H.K. et al. In-depth analysis of site-specific N-glycosylation in vitronectin from human plasma by tandem mass spectrometry with immunoprecipitation. Anal Bioanal Chem 406, 7999–8011 (2014). https://doi.org/10.1007/s00216-014-8226-5

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