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

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• 作者：Martin Pabst ; Iva Bene?ová ; Stephan R. Fagerer ; Mathias Jacobsen ; Klaus Eyer ; Gregor Schmidt ; Robert Steinhoff ; Jasmin Krismer ; Fabian Wahl ; Jan Preisler ; Renato Zenobi
• 刊名：Journal of Proteome Research
• 出版年：2016
• 出版时间：January 4, 2016
• 年：2016
• 卷：15
• 期：1
• 页码：326-331
• 全文大小：323K
• ISSN：1535-3907

文摘

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.