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J Proteome Res. 2016 Jan 4;15(1):326-31. doi: 10.1021/acs.jproteome.5b00899. Epub 2015 Nov 25.

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

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Department of Chemistry and Applied Biosciences, ETH Zurich , 8093 Zurich, Switzerland.
Department of Chemistry, Masaryk University , 625 00 Brno, Czech Republic.
Department of Biosystems Science and Engineering (D-BSSE), ETH Zurich , 4058 Basel, Switzerland.
Sigma-Aldrich Chemie GmbH , 9470 Buchs, Switzerland.
Central European Institute of Technology (CEITEC), Masaryk University , 625 00 Brno, Czech Republic.


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.


IgG; LC−ESI−MS; MALDI MS; glycopeptides; stable isotope labeling

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