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Identification of N-glycosylation sites of the murine neural cell adhesion molecule NCAM by MALDI-TOF and MALDI-FTICR mass spectrometry

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Abstract

Mass spectrometry has been shown in recent years to be a powerful tool to determine accurate molecular masses and sequences of peptides and proteins and post-translational modifications such as glycosylation, phosphorylation, and sulfation. For glycosylation, it has been increasingly recognized to be of pivotal importance to identify whether potential glycosylation sites are actually modified by glycans, because functions of proteins may be modulated or depend on the presence of glycans at specific sites. Several recent reports have established that mass spectrometric techniques such as matrix-assisted laser desorption/ionization or electrospray ionization mass spectrometry (MALDI-TOF or ESI-MS, respectively) with or without preceding HPLC and in combination with PNGase F treatment are suited to analyze whether consensus sequences for N-glycosylation are glycosylated or not. Here we report the mass spectrometric analysis of the six potential N-glycosylation sites of the neural cell adhesion molecule NCAM from adult mouse brain. Unmodified peptides and glycopeptides each carrying a single glycosylation site were generated from NCAM by AspN and trypsin treatment and submitted to reversed-phase HPLC with or without prior enzymatic release of N-glycans. The resulting peptides were analyzed by MALDI-TOF-MS. In addition, high-resolution Fourier transform–ion cyclotron resonance (MALDI-FTICR) mass spectrometry was performed after in-gel deglycosylation and subsequent trypsin digestion. By using these procedures all six consensus sequences were shown to be glycosylated; the observation of an unmodified peptide with the consensus sequence N-1 indicates only partial glycosylation at this site.

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Abbreviations

amu:

atomic mass units

AspN:

endoproteinase AspN

CAM:

cell adhesion molecule

ESI:

electrospray ionization

FTICR:

Fourier transform–ion cyclotron resonance

IgSF:

immunoglobulin superfamily

MALDI-TOF:

matrix-assisted laser desorption ionization–time of flight

MS:

mass spectrometry

NCAM:

neural cell adhesion molecule

PNGase F:

peptide-N 4-(N-acetyl-β-glucosaminyl)asparagine amidase

PSA:

polysialic acid

TFA:

trifluoroacetic acid

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Acknowledgements

We thank Bernhard Gehrig for the preparation of NCAM for the MALDI-FTICR-MS analysis, and Ursula Munzel for expert secretarial assistance. The expert assistance of Nikolay Youhnovski with the FTICR-MS is gratefully acknowledged. This work has been supported by grants from the Deutsche Forschungsgemeinschaft (SFB 284/A8, B.S., and Biopolymer-MS/Pr-175/4, M.P.).

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Authors and Affiliations

  1. Institute for Physiology, Biochemistry and Animal Health, University of Bonn, Katzenburgweg 7–9, 53115, Bonn, Germany

    Claus Albach & Brigitte Schmitz

  2. Department of Chemistry, Laboratory of Analytical Chemistry, University of Konstanz, 78457, Konstanz, Germany

    Eugen Damoc, Thomas Denzinger & Michael Przybylski

  3. Center for Molecular Neurobiology, University of Hamburg, Martinistr. 52, 20246, Hamburg, Germany

    Melitta Schachner

Authors
  1. Claus Albach
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  2. Eugen Damoc
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  3. Thomas Denzinger
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  4. Melitta Schachner
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  5. Michael Przybylski
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  6. Brigitte Schmitz
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Correspondence to Brigitte Schmitz.

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Albach, C., Damoc, E., Denzinger, T. et al. Identification of N-glycosylation sites of the murine neural cell adhesion molecule NCAM by MALDI-TOF and MALDI-FTICR mass spectrometry. Anal Bioanal Chem 378, 1129–1135 (2004). https://doi.org/10.1007/s00216-003-2383-2

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  • DOI: https://doi.org/10.1007/s00216-003-2383-2

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