Lectin-based enrichment method for glycoproteomics using hollow fiber flow field-flow fractionation: Application to streptococcus pyogenes

Dukjin Kang, Eun Sun Ji, Myeong Hee Moon, Jong Shin Yoo

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

This paper presents a new application of hollow fiber flow field-flow fractionation (HF5) as a preparative method to preconcentrate high mannose type N-linked glycoproteins from Streptococcus pyogenes by means of the mannose-specific binding affinity between concanavalian A (ConA) and N-linked glycosylated proteins. Prior to fractionation of N-linked glycoproteins from bacterial lysates, it was examined that ConA formed several types of multimers depending on the pH values (4, 6, and 8) of the carrier solution and it was confirmed that the molecular weight (MW) of ConA, spiked with R-1 acid glycoprotein (AGP) as a standard glycoprotein, increased due to binding with the mannose moiety of AGP. After adding ConA to bacterial lysates, mannose type N-linked glycoproteins were found to be enriched when the ConA fraction was isolated from whole bacterial lysates through HF5 run. For the identification of glycoproteins, the ConA fraction of HF5 was tryptically digested and followed by twodimensional nanoflow strong cation exchange-reversed phase liquid chromatography-electrospray ionization-tandem mass spectrometry (2D SCX-RPLC-ESI-MS-MS) analysis to identify the N-linked glycoprotein species. From two-dimensional shotgun analyses, 45 proteins that exist on the Asn-Xaa-Ser/Thr sequence were identified as high mannose type N-linked glycoprotein. As a result, it was first demonstrated that HF5 is an alternative tool to enrich high mannose type N-linked glycoproteins using ConA-specific binding affinity.

Original languageEnglish
Pages (from-to)2855-2862
Number of pages8
JournalJournal of Proteome Research
Volume9
Issue number6
DOIs
Publication statusPublished - 2010 Jun 4

Fingerprint

Field Flow Fractionation
Streptococcus pyogenes
Fractionation
Lectins
Flow fields
Glycoproteins
Mannose
Fibers
Electrospray ionization
Acids
Electrospray Ionization Mass Spectrometry
Liquid chromatography
Firearms
Reverse-Phase Chromatography
Tandem Mass Spectrometry
Mass spectrometry
Cations
Proteins

All Science Journal Classification (ASJC) codes

  • Chemistry(all)
  • Biochemistry

Cite this

@article{2d05ba90c2b64ed9ac7e0a48262f7cd7,
title = "Lectin-based enrichment method for glycoproteomics using hollow fiber flow field-flow fractionation: Application to streptococcus pyogenes",
abstract = "This paper presents a new application of hollow fiber flow field-flow fractionation (HF5) as a preparative method to preconcentrate high mannose type N-linked glycoproteins from Streptococcus pyogenes by means of the mannose-specific binding affinity between concanavalian A (ConA) and N-linked glycosylated proteins. Prior to fractionation of N-linked glycoproteins from bacterial lysates, it was examined that ConA formed several types of multimers depending on the pH values (4, 6, and 8) of the carrier solution and it was confirmed that the molecular weight (MW) of ConA, spiked with R-1 acid glycoprotein (AGP) as a standard glycoprotein, increased due to binding with the mannose moiety of AGP. After adding ConA to bacterial lysates, mannose type N-linked glycoproteins were found to be enriched when the ConA fraction was isolated from whole bacterial lysates through HF5 run. For the identification of glycoproteins, the ConA fraction of HF5 was tryptically digested and followed by twodimensional nanoflow strong cation exchange-reversed phase liquid chromatography-electrospray ionization-tandem mass spectrometry (2D SCX-RPLC-ESI-MS-MS) analysis to identify the N-linked glycoprotein species. From two-dimensional shotgun analyses, 45 proteins that exist on the Asn-Xaa-Ser/Thr sequence were identified as high mannose type N-linked glycoprotein. As a result, it was first demonstrated that HF5 is an alternative tool to enrich high mannose type N-linked glycoproteins using ConA-specific binding affinity.",
author = "Dukjin Kang and Ji, {Eun Sun} and Moon, {Myeong Hee} and Yoo, {Jong Shin}",
year = "2010",
month = "6",
day = "4",
doi = "10.1021/pr900937w",
language = "English",
volume = "9",
pages = "2855--2862",
journal = "Journal of Proteome Research",
issn = "1535-3893",
publisher = "American Chemical Society",
number = "6",

}

Lectin-based enrichment method for glycoproteomics using hollow fiber flow field-flow fractionation : Application to streptococcus pyogenes. / Kang, Dukjin; Ji, Eun Sun; Moon, Myeong Hee; Yoo, Jong Shin.

In: Journal of Proteome Research, Vol. 9, No. 6, 04.06.2010, p. 2855-2862.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Lectin-based enrichment method for glycoproteomics using hollow fiber flow field-flow fractionation

T2 - Application to streptococcus pyogenes

AU - Kang, Dukjin

AU - Ji, Eun Sun

AU - Moon, Myeong Hee

AU - Yoo, Jong Shin

PY - 2010/6/4

Y1 - 2010/6/4

N2 - This paper presents a new application of hollow fiber flow field-flow fractionation (HF5) as a preparative method to preconcentrate high mannose type N-linked glycoproteins from Streptococcus pyogenes by means of the mannose-specific binding affinity between concanavalian A (ConA) and N-linked glycosylated proteins. Prior to fractionation of N-linked glycoproteins from bacterial lysates, it was examined that ConA formed several types of multimers depending on the pH values (4, 6, and 8) of the carrier solution and it was confirmed that the molecular weight (MW) of ConA, spiked with R-1 acid glycoprotein (AGP) as a standard glycoprotein, increased due to binding with the mannose moiety of AGP. After adding ConA to bacterial lysates, mannose type N-linked glycoproteins were found to be enriched when the ConA fraction was isolated from whole bacterial lysates through HF5 run. For the identification of glycoproteins, the ConA fraction of HF5 was tryptically digested and followed by twodimensional nanoflow strong cation exchange-reversed phase liquid chromatography-electrospray ionization-tandem mass spectrometry (2D SCX-RPLC-ESI-MS-MS) analysis to identify the N-linked glycoprotein species. From two-dimensional shotgun analyses, 45 proteins that exist on the Asn-Xaa-Ser/Thr sequence were identified as high mannose type N-linked glycoprotein. As a result, it was first demonstrated that HF5 is an alternative tool to enrich high mannose type N-linked glycoproteins using ConA-specific binding affinity.

AB - This paper presents a new application of hollow fiber flow field-flow fractionation (HF5) as a preparative method to preconcentrate high mannose type N-linked glycoproteins from Streptococcus pyogenes by means of the mannose-specific binding affinity between concanavalian A (ConA) and N-linked glycosylated proteins. Prior to fractionation of N-linked glycoproteins from bacterial lysates, it was examined that ConA formed several types of multimers depending on the pH values (4, 6, and 8) of the carrier solution and it was confirmed that the molecular weight (MW) of ConA, spiked with R-1 acid glycoprotein (AGP) as a standard glycoprotein, increased due to binding with the mannose moiety of AGP. After adding ConA to bacterial lysates, mannose type N-linked glycoproteins were found to be enriched when the ConA fraction was isolated from whole bacterial lysates through HF5 run. For the identification of glycoproteins, the ConA fraction of HF5 was tryptically digested and followed by twodimensional nanoflow strong cation exchange-reversed phase liquid chromatography-electrospray ionization-tandem mass spectrometry (2D SCX-RPLC-ESI-MS-MS) analysis to identify the N-linked glycoprotein species. From two-dimensional shotgun analyses, 45 proteins that exist on the Asn-Xaa-Ser/Thr sequence were identified as high mannose type N-linked glycoprotein. As a result, it was first demonstrated that HF5 is an alternative tool to enrich high mannose type N-linked glycoproteins using ConA-specific binding affinity.

UR - http://www.scopus.com/inward/record.url?scp=77954618580&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77954618580&partnerID=8YFLogxK

U2 - 10.1021/pr900937w

DO - 10.1021/pr900937w

M3 - Article

C2 - 20377246

AN - SCOPUS:77954618580

VL - 9

SP - 2855

EP - 2862

JO - Journal of Proteome Research

JF - Journal of Proteome Research

SN - 1535-3893

IS - 6

ER -