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

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Authors

PABST Martin BENEŠOVÁ Iva FAGERER Stephan R JACOBSEN Mathias EYER Klaus SCHMIDT Gregor STEINHOFF Robert KRISMER Jasmin WAHL Fabian PREISLER Jan ZENOBI Renato

Year of publication 2016
Type Article in Periodical
Magazine / Source Journal of Proteome Research
MU Faculty or unit

Faculty of Science

Citation
Doi http://dx.doi.org/10.1021/acs.jproteome.5b00899
Field Analytic chemistry
Keywords glycopeptides; stable isotope labeling; IgG; MALDI MS; LC-ESI-MS
Description 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 ((D4C4)-C-13) provides an 8 Da mass increment over the light natural form ((H4C4)-C-12), 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 IgGI Pc glycopeptides from polyclonal IgG samples with respect to their galactosylation and sialylation patterns using MALDI MS and LC-ESI-MS.
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