Differential Isotope Labeling of Glycopeptides for Accurate Determination of Differences in Site-Specific Glycosylation
Language English Country United States Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
- Keywords
- IgG, LC−ESI−MS, MALDI MS, glycopeptides, stable isotope labeling,
- MeSH
- Succinic Anhydrides chemistry MeSH
- Glycopeptides chemistry MeSH
- Glycosylation MeSH
- Spectrometry, Mass, Electrospray Ionization MeSH
- Immunoglobulin G chemistry MeSH
- Isotope Labeling MeSH
- Humans MeSH
- Protein Processing, Post-Translational * MeSH
- Proteomics methods MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Succinic Anhydrides MeSH
- Glycopeptides MeSH
- Immunoglobulin G MeSH
- succinic anhydride MeSH Browser
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 (D4(13)C4) provides an 8 Da mass increment over the light natural form (H4(12)C4), 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.
Central European Institute of Technology Masaryk University 625 00 Brno Czech Republic
Department of Biosystems Science and Engineering ETH Zurich 4058 Basel Switzerland
Department of Chemistry and Applied Biosciences ETH Zurich 8093 Zurich Switzerland
Department of Chemistry Masaryk University 625 00 Brno Czech Republic
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