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J Biomol Tech. 2010 September; 21(3 Suppl): S34–S35.
PMCID: PMC2918219

N-glycosylation of Antibodies Characterized by Complementary Top-Down ESI and Bottom-Up MALDI Mass Spectrometric Techniques

D. Wunderlich, M. Macht, and P.O. Schmit
Bruker Daltonik GmbH, Bremen, Germany



Antibodies represent one of the most important classes of glycoproteins playing a central role in the immune response of living organisms. Furthermore, there is a growing interest in recombinant antibodies as potential biotherapeutic agents. The analysis of the N-glycosylation pattern present on antibodies is challenging due to its heterogeneous structure. The glycan profile is highly dependent on the process by which a recombinant glycoprotein is generated, such as host organism and growth conditions. Changes to the glycosylation pattern can significantly alter biological function. To characterize the N-glycosylation pattern, various mass spectrometric techniques may be applied to analyze either the intact glycoprotein or the N-glycopeptides obtained from enzymatic digestion. We describe here the in-depth characterization of the N-glycosylation pattern in various antibodies by utilizing two complementary MS based techniques: I) fast top-down analysis by LC-MS accurate mass measurement of the intact antibody and the released heavy chain performed on a next generation ESI-ultrahigh-resolution (UHR)-TOF instrument; II) comprehensive bottom-up characterization of the N-glycosylation pattern by nanoLC-MALDI-TOF/TOF analysis of N-glycopeptides obtained from digested antibodies. Fast LC-ESI-UHR-TOF analysis provides high-resolution, high-mass accuracy (confident low ppm) top-down data enabling a rapid assignment of the major N-glycosylation isoforms. As a complementary bottom-up technique, LC-MALDI-TOF/TOF analysis of tryptic antibody digests provides a detail-rich picture of the highly complex pattern of N-linked glycans in form of the respective N-glycopeptides. As a unique feature, MALDI-TOF/TOF data generated from N-glycopeptides provides information on both the peptide sequence and the structure of the N-linked glycan moiety in one and the same spectrum. Selective screening and subsequent analysis of MS/MS spectra of N-glycopeptides from large LC-MALDI-MS/MS datasets is facilitated by dedicated, newly implemented software features.

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