title: Differential Isotope Labeling of Glycopeptides for Accurate Determination of Differences in Site-Specific Glycosylation.
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 (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.
authors.affiliation: Department of Chemistry and Applied Biosciences, ETH Zurich , 8093 Zurich, Switzerland.
authors.forename: Martin
authors.initials: M
authors.lastname: Pabst
authors.affiliation: Department of Chemistry and Applied Biosciences, ETH Zurich , 8093 Zurich, Switzerland.|Department of Chemistry, Masaryk University , 625 00 Brno, Czech Republic.
authors.forename: Iva
authors.initials: I
authors.lastname: Benešová
authors.affiliation: Department of Chemistry and Applied Biosciences, ETH Zurich , 8093 Zurich, Switzerland.
authors.forename: Stephan R
authors.initials: SR
authors.lastname: Fagerer
authors.affiliation: Department of Chemistry and Applied Biosciences, ETH Zurich , 8093 Zurich, Switzerland.
authors.forename: Mathias
authors.initials: M
authors.lastname: Jacobsen
authors.affiliation: Department of Chemistry and Applied Biosciences, ETH Zurich , 8093 Zurich, Switzerland.
authors.forename: Klaus
authors.initials: K
authors.lastname: Eyer
authors.affiliation: Department of Biosystems Science and Engineering (D-BSSE), ETH Zurich , 4058 Basel, Switzerland.
authors.forename: Gregor
authors.initials: G
authors.lastname: Schmidt
authors.affiliation: Department of Chemistry and Applied Biosciences, ETH Zurich , 8093 Zurich, Switzerland.
authors.forename: Robert
authors.initials: R
authors.lastname: Steinhoff
authors.affiliation: Department of Chemistry and Applied Biosciences, ETH Zurich , 8093 Zurich, Switzerland.
authors.forename: Jasmin
authors.initials: J
authors.lastname: Krismer
authors.affiliation: Sigma-Aldrich Chemie GmbH , 9470 Buchs, Switzerland.
authors.forename: Fabian
authors.initials: F
authors.lastname: Wahl
authors.affiliation: Department of Chemistry, Masaryk University , 625 00 Brno, Czech Republic.|Central European Institute of Technology (CEITEC), Masaryk University , 625 00 Brno, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Preisler
authors.affiliation: Department of Chemistry and Applied Biosciences, ETH Zurich , 8093 Zurich, Switzerland.
authors.forename: Renato
authors.initials: R
authors.lastname: Zenobi
mesh_terms: D006020:Glycopeptides / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D007553:Isotope Labeling; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013387:Succinic Anhydrides / Q000737:chemistry
chemical_list: D006020:Glycopeptides; D007074:Immunoglobulin G; D013387:Succinic Anhydrides; C031801:succinic anhydride
keywords: IgG; LC−ESI−MS; MALDI MS; glycopeptides; stable isotope labeling