Characterization of Site-Specific N-Glycosylation

Helga Hevér, Z. Darula, Katalin F. Medzihradszky

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Even if a consensus sequence has been identified for a posttranslational modification, the presence of such a sequence motif only indicates the possibility, not the certainty that the modification actually occurs. Proteins can be glycosylated on certain amino acid side chains, and these modifications are designated as C-, N-, and O-glycosylation. C-mannosylation occurs on Trp residues within a relatively loosely defined consensus motif. N-glycosylated species are modified at Asn residues of Asn-Xxx-Ser/Thr/Cys sequons (where Xxx can be any amino acid except proline). N-linked oligosaccharides share a common core structure of GlcNAc2Man3. In addition, an enzyme, peptide N-glycosidase F (PNGase F), removes most of the common N-linked carbohydrates unaltered from proteins while hydrolyzing the originally glycosylated Asn residue to Asp. O-glycosylation occurs at Ser, Thr, and Tyr residues, usually in sequence stretches rich in hydroxy-amino acids. O-glycosylation lacks a common core structure. Mammalian proteins have been reported bearing O-linked N-acetylgalactosamine, fucose, glucose, xylose, mannose, and corresponding elongated structures, as well as N-acetylglucosamine. Chemical methods are used to liberate these oligosaccharides because no enzyme would remove all the different O-linked carbohydrates. Characterization of both N- and O-glycosylation is complicated by the fact that the same positions within a population of protein molecules may feature an array of different carbohydrate structures, or remain unmodified. This site-specific heterogeneity may vary by species and tissue, and may also be affected by physiological changes. For addressing site-specific carbohydrate heterogeneity mass spectrometry has become the method of choice. Reversed-phase HPLC directly coupled with electrospray ionization mass spectrometry (LC/ESI-MS/MS) offers the best solution. Using a mass spectrometer as online detector not only assures the analysis of every component eluting (mass mapping), but also at the same time diagnostic carbohydrate ions can be generated by collisional activation that permits the selective and specific detection of glycopeptides. In addition, ESI-compatible alternative MS/MS techniques, electron-capture and electron-transfer dissociation, aid glycopeptide identification as well as modification site assignments.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages93-125
Number of pages33
DOIs
Publication statusPublished - Jan 1 2019

Publication series

NameMethods in Molecular Biology
Volume1934
ISSN (Print)1064-3745

Fingerprint

Glycosylation
Carbohydrates
Glycopeptides
Oligosaccharides
Amino Acids
Proteins
Electrons
Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
Acetylgalactosamine
Hydroxy Acids
Fucose
Acetylglucosamine
Electrospray Ionization Mass Spectrometry
Consensus Sequence
Enzymes
Post Translational Protein Processing
Mannose
Proline
Mass Spectrometry
High Pressure Liquid Chromatography

Keywords

  • CID
  • Electrospray ionization mass spectrometry
  • ETD
  • HCD
  • ISCID
  • N-glycosylation
  • nLC/ESI-MS/MS
  • Site-specific glycosylation

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Hevér, H., Darula, Z., & Medzihradszky, K. F. (2019). Characterization of Site-Specific N-Glycosylation. In Methods in Molecular Biology (pp. 93-125). (Methods in Molecular Biology; Vol. 1934). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-9055-9_8

Characterization of Site-Specific N-Glycosylation. / Hevér, Helga; Darula, Z.; Medzihradszky, Katalin F.

Methods in Molecular Biology. Humana Press Inc., 2019. p. 93-125 (Methods in Molecular Biology; Vol. 1934).

Research output: Chapter in Book/Report/Conference proceedingChapter

Hevér, H, Darula, Z & Medzihradszky, KF 2019, Characterization of Site-Specific N-Glycosylation. in Methods in Molecular Biology. Methods in Molecular Biology, vol. 1934, Humana Press Inc., pp. 93-125. https://doi.org/10.1007/978-1-4939-9055-9_8
Hevér H, Darula Z, Medzihradszky KF. Characterization of Site-Specific N-Glycosylation. In Methods in Molecular Biology. Humana Press Inc. 2019. p. 93-125. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-9055-9_8
Hevér, Helga ; Darula, Z. ; Medzihradszky, Katalin F. / Characterization of Site-Specific N-Glycosylation. Methods in Molecular Biology. Humana Press Inc., 2019. pp. 93-125 (Methods in Molecular Biology).
@inbook{39a1a110fef849e5acfca076368755df,
title = "Characterization of Site-Specific N-Glycosylation",
abstract = "Even if a consensus sequence has been identified for a posttranslational modification, the presence of such a sequence motif only indicates the possibility, not the certainty that the modification actually occurs. Proteins can be glycosylated on certain amino acid side chains, and these modifications are designated as C-, N-, and O-glycosylation. C-mannosylation occurs on Trp residues within a relatively loosely defined consensus motif. N-glycosylated species are modified at Asn residues of Asn-Xxx-Ser/Thr/Cys sequons (where Xxx can be any amino acid except proline). N-linked oligosaccharides share a common core structure of GlcNAc2Man3. In addition, an enzyme, peptide N-glycosidase F (PNGase F), removes most of the common N-linked carbohydrates unaltered from proteins while hydrolyzing the originally glycosylated Asn residue to Asp. O-glycosylation occurs at Ser, Thr, and Tyr residues, usually in sequence stretches rich in hydroxy-amino acids. O-glycosylation lacks a common core structure. Mammalian proteins have been reported bearing O-linked N-acetylgalactosamine, fucose, glucose, xylose, mannose, and corresponding elongated structures, as well as N-acetylglucosamine. Chemical methods are used to liberate these oligosaccharides because no enzyme would remove all the different O-linked carbohydrates. Characterization of both N- and O-glycosylation is complicated by the fact that the same positions within a population of protein molecules may feature an array of different carbohydrate structures, or remain unmodified. This site-specific heterogeneity may vary by species and tissue, and may also be affected by physiological changes. For addressing site-specific carbohydrate heterogeneity mass spectrometry has become the method of choice. Reversed-phase HPLC directly coupled with electrospray ionization mass spectrometry (LC/ESI-MS/MS) offers the best solution. Using a mass spectrometer as online detector not only assures the analysis of every component eluting (mass mapping), but also at the same time diagnostic carbohydrate ions can be generated by collisional activation that permits the selective and specific detection of glycopeptides. In addition, ESI-compatible alternative MS/MS techniques, electron-capture and electron-transfer dissociation, aid glycopeptide identification as well as modification site assignments.",
keywords = "CID, Electrospray ionization mass spectrometry, ETD, HCD, ISCID, N-glycosylation, nLC/ESI-MS/MS, Site-specific glycosylation",
author = "Helga Hev{\'e}r and Z. Darula and Medzihradszky, {Katalin F.}",
year = "2019",
month = "1",
day = "1",
doi = "10.1007/978-1-4939-9055-9_8",
language = "English",
series = "Methods in Molecular Biology",
publisher = "Humana Press Inc.",
pages = "93--125",
booktitle = "Methods in Molecular Biology",

}

TY - CHAP

T1 - Characterization of Site-Specific N-Glycosylation

AU - Hevér, Helga

AU - Darula, Z.

AU - Medzihradszky, Katalin F.

PY - 2019/1/1

Y1 - 2019/1/1

N2 - Even if a consensus sequence has been identified for a posttranslational modification, the presence of such a sequence motif only indicates the possibility, not the certainty that the modification actually occurs. Proteins can be glycosylated on certain amino acid side chains, and these modifications are designated as C-, N-, and O-glycosylation. C-mannosylation occurs on Trp residues within a relatively loosely defined consensus motif. N-glycosylated species are modified at Asn residues of Asn-Xxx-Ser/Thr/Cys sequons (where Xxx can be any amino acid except proline). N-linked oligosaccharides share a common core structure of GlcNAc2Man3. In addition, an enzyme, peptide N-glycosidase F (PNGase F), removes most of the common N-linked carbohydrates unaltered from proteins while hydrolyzing the originally glycosylated Asn residue to Asp. O-glycosylation occurs at Ser, Thr, and Tyr residues, usually in sequence stretches rich in hydroxy-amino acids. O-glycosylation lacks a common core structure. Mammalian proteins have been reported bearing O-linked N-acetylgalactosamine, fucose, glucose, xylose, mannose, and corresponding elongated structures, as well as N-acetylglucosamine. Chemical methods are used to liberate these oligosaccharides because no enzyme would remove all the different O-linked carbohydrates. Characterization of both N- and O-glycosylation is complicated by the fact that the same positions within a population of protein molecules may feature an array of different carbohydrate structures, or remain unmodified. This site-specific heterogeneity may vary by species and tissue, and may also be affected by physiological changes. For addressing site-specific carbohydrate heterogeneity mass spectrometry has become the method of choice. Reversed-phase HPLC directly coupled with electrospray ionization mass spectrometry (LC/ESI-MS/MS) offers the best solution. Using a mass spectrometer as online detector not only assures the analysis of every component eluting (mass mapping), but also at the same time diagnostic carbohydrate ions can be generated by collisional activation that permits the selective and specific detection of glycopeptides. In addition, ESI-compatible alternative MS/MS techniques, electron-capture and electron-transfer dissociation, aid glycopeptide identification as well as modification site assignments.

AB - Even if a consensus sequence has been identified for a posttranslational modification, the presence of such a sequence motif only indicates the possibility, not the certainty that the modification actually occurs. Proteins can be glycosylated on certain amino acid side chains, and these modifications are designated as C-, N-, and O-glycosylation. C-mannosylation occurs on Trp residues within a relatively loosely defined consensus motif. N-glycosylated species are modified at Asn residues of Asn-Xxx-Ser/Thr/Cys sequons (where Xxx can be any amino acid except proline). N-linked oligosaccharides share a common core structure of GlcNAc2Man3. In addition, an enzyme, peptide N-glycosidase F (PNGase F), removes most of the common N-linked carbohydrates unaltered from proteins while hydrolyzing the originally glycosylated Asn residue to Asp. O-glycosylation occurs at Ser, Thr, and Tyr residues, usually in sequence stretches rich in hydroxy-amino acids. O-glycosylation lacks a common core structure. Mammalian proteins have been reported bearing O-linked N-acetylgalactosamine, fucose, glucose, xylose, mannose, and corresponding elongated structures, as well as N-acetylglucosamine. Chemical methods are used to liberate these oligosaccharides because no enzyme would remove all the different O-linked carbohydrates. Characterization of both N- and O-glycosylation is complicated by the fact that the same positions within a population of protein molecules may feature an array of different carbohydrate structures, or remain unmodified. This site-specific heterogeneity may vary by species and tissue, and may also be affected by physiological changes. For addressing site-specific carbohydrate heterogeneity mass spectrometry has become the method of choice. Reversed-phase HPLC directly coupled with electrospray ionization mass spectrometry (LC/ESI-MS/MS) offers the best solution. Using a mass spectrometer as online detector not only assures the analysis of every component eluting (mass mapping), but also at the same time diagnostic carbohydrate ions can be generated by collisional activation that permits the selective and specific detection of glycopeptides. In addition, ESI-compatible alternative MS/MS techniques, electron-capture and electron-transfer dissociation, aid glycopeptide identification as well as modification site assignments.

KW - CID

KW - Electrospray ionization mass spectrometry

KW - ETD

KW - HCD

KW - ISCID

KW - N-glycosylation

KW - nLC/ESI-MS/MS

KW - Site-specific glycosylation

UR - http://www.scopus.com/inward/record.url?scp=85068105001&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85068105001&partnerID=8YFLogxK

U2 - 10.1007/978-1-4939-9055-9_8

DO - 10.1007/978-1-4939-9055-9_8

M3 - Chapter

C2 - 31256376

AN - SCOPUS:85068105001

T3 - Methods in Molecular Biology

SP - 93

EP - 125

BT - Methods in Molecular Biology

PB - Humana Press Inc.

ER -