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Quantitation of Glycopeptides by ESI/MS - size of the peptide part strongly affects the relative proportions and allows discovery of new glycan compositions of Ceruloplasmin

  • Melissa Baerenfaenger
  • Manuela Moritz
  • Bernd MeyerEmail author
Original Article

Abstract

Significant changes of glycan structures are observed in humans if diseases like cancer, arthritis or inflammation are present. Thus, interest in biomarkers based on glycan structures has rapidly emerged in recent years and monitoring disease specific changes of glycosylation and their quantification is of great interest. Mass spectrometry is most commonly used to characterize and quantify glycopeptides and glycans liberated from the glycoprotein of interest. However, ionization properties of glycopeptides can strongly depend on their composition and can therefore lead to intensities that do not reflect the actual proportions present in the intact glycoprotein. Here we show that an increase in the length of the peptide can lead to a more accurate determination and quantification of the glycans. The four glycosylation sites of human serum ceruloplasmin from 17 different individuals were analyzed using glycopeptides of varying peptide lengths, obtained by action of different proteases and by limited digestion. In most cases, highly sialylated compositions showed an increased relative abundance with increasing peptide length. We observed a relative increase of triantennary glycans of up to a factor of three and, even more, MS peaks corresponding to tetraantennary compositions on ceruloplasmin at glycosite 137N in all 17 samples, which we did not detect using a bottom up approach. The data presented here leads to the conclusion that a middle down - or when possible a top down - approach is favorable for qualitative and quantitative analysis of the glycosylation of glycoproteins.

Keywords

Glycan quantification Glycopeptide analysis Middle down approach Ceruloplasmin 

Abbreviations

H

hexose

N

N-acetylhexosamine

S

sialic acid

F

fucose

Notes

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Supplementary material

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10719_2018_9852_MOESM3_ESM.pdf (2.7 mb)
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Authors and Affiliations

  1. 1.Organic Chemistry, Department of ChemistryUniversity of HamburgHamburgGermany

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