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

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

Research output: Contribution to journalArticle

18 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)7999-8011
Number of pages13
JournalAnalytical and Bioanalytical Chemistry
Volume406
Issue number30
DOIs
Publication statusPublished - 2014 Jan 1

Fingerprint

Plasma (human)
Glycosylation
Vitronectin
Glycopeptides
Tandem Mass Spectrometry
Immunoprecipitation
Mass spectrometry
Liquid chromatography
Ions
Hydrophobic and Hydrophilic Interactions
Liquid Chromatography
Hepatocellular Carcinoma
Glycoproteins
Cytology
Extracellular Matrix
Cell Biology
Blood
Monoclonal Antibodies
Plasmas
Peptides

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Biochemistry

Cite this

Hwang, Heeyoun ; Lee, Ju Yeon ; Lee, Hyun Kyoung ; Park, Gun Wook ; Jeong, Hoi Keun ; Moon, Myeong Hee ; Kim, Jin Young ; Yoo, Jong Shin. / In-depth analysis of site-specific N-glycosylation in vitronectin from human plasma by tandem mass spectrometry with immunoprecipitation. In: Analytical and Bioanalytical Chemistry. 2014 ; Vol. 406, No. 30. pp. 7999-8011.
@article{1271792d49394a2b896b54528b2c773a,
title = "In-depth analysis of site-specific N-glycosylation in vitronectin from human plasma by tandem mass spectrometry with immunoprecipitation",
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.",
author = "Heeyoun Hwang and Lee, {Ju Yeon} and Lee, {Hyun Kyoung} and Park, {Gun Wook} and Jeong, {Hoi Keun} and Moon, {Myeong Hee} and Kim, {Jin Young} and Yoo, {Jong Shin}",
year = "2014",
month = "1",
day = "1",
doi = "10.1007/s00216-014-8226-5",
language = "English",
volume = "406",
pages = "7999--8011",
journal = "Fresenius Zeitschrift fur Analytische Chemie",
issn = "0016-1152",
publisher = "Springer Verlag",
number = "30",

}

In-depth analysis of site-specific N-glycosylation in vitronectin from human plasma by tandem mass spectrometry with immunoprecipitation. / Hwang, Heeyoun; Lee, Ju Yeon; Lee, Hyun Kyoung; Park, Gun Wook; Jeong, Hoi Keun; Moon, Myeong Hee; Kim, Jin Young; Yoo, Jong Shin.

In: Analytical and Bioanalytical Chemistry, Vol. 406, No. 30, 01.01.2014, p. 7999-8011.

Research output: Contribution to journalArticle

TY - JOUR

T1 - In-depth analysis of site-specific N-glycosylation in vitronectin from human plasma by tandem mass spectrometry with immunoprecipitation

AU - Hwang, Heeyoun

AU - Lee, Ju Yeon

AU - Lee, Hyun Kyoung

AU - Park, Gun Wook

AU - Jeong, Hoi Keun

AU - Moon, Myeong Hee

AU - Kim, Jin Young

AU - Yoo, Jong Shin

PY - 2014/1/1

Y1 - 2014/1/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84914152394&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84914152394&partnerID=8YFLogxK

U2 - 10.1007/s00216-014-8226-5

DO - 10.1007/s00216-014-8226-5

M3 - Article

VL - 406

SP - 7999

EP - 8011

JO - Fresenius Zeitschrift fur Analytische Chemie

JF - Fresenius Zeitschrift fur Analytische Chemie

SN - 0016-1152

IS - 30

ER -