Differential Isotope Labeling of Glycopeptides for Accurate Determination of Differences in Site-Specific Glycosylation

Martin Pabst, Iva Benešová, Stephan R. Fagerer, Mathias Jacobsen, Klaus Eyer, Gregor Schmidt, Robert Steinhoff, Jasmin Krismer, Fabian Wahl, More Authors

Research output: Contribution to journalArticleScientificpeer-review

12 Citations (Scopus)

Abstract

We introduce a stable isotope labeling approach for glycopeptides that allows a specific glycosylation site in a protein to be quantitatively evaluated using mass spectrometry. Succinic anhydride is used to specifically label primary amino groups of the peptide portion of the glycopeptides. The heavy form (D413C4) provides an 8 Da mass increment over the light natural form (H412C4), allowing simultaneous analysis and direct comparison of two glycopeptide profiles in a single MS scan. We have optimized a protocol for an in-solution trypsin digestion, a one-pot labeling procedure, and a post-labeling solid-phase extraction to obtain purified and labeled glycopeptides. We provide the first demonstration of this approach by comparing IgG1 Fc glycopeptides from polyclonal IgG samples with respect to their galactosylation and sialylation patterns using MALDI MS and LC-ESI-MS.

Original languageEnglish
Pages (from-to)326-331
JournalJournal of Proteome Research
Volume15
Issue number1
DOIs
Publication statusPublished - 2016
Externally publishedYes

Keywords

  • glycopeptides
  • IgG
  • LC-ESI-MS
  • MALDI MS
  • stable isotope labeling

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