Characterization of the N-glycosylation Pattern of Antibodies by ESI – and MALDI Mass Spectrometry

Analysis of the N-glycosylation pattern on antibodies is described using complementary mass spectrometric strategies based on both, top-down ESI-UHR-TOF and bottom-up LC-MALDI-TOF/TOF. Fast LC-ESI-UHR-TOF analysis, performed on the Bruker maXisTM, provides highresolution, high-mass accuracy (confident low ppm) data for both, intact antibodies and released antibody heavy chains allowing a rapid assignment of the major N-glycosylation isoforms. Bruker ́s new ultrafleXtremeTM platform facilitates in-depth characterization of the antibodies ́ N-glycosylation patterns via LC-MALDI-TOF/TOF analysis of the N-glycopeptides generated from digested antibodies. The improved resolution provided by the instrument delivers an extremely detailed picture of the highly complex patterns of N-linked glycans present on antibodies from different origins. As a unique feature, MALDI-TOF/TOF data generated from N-glycopeptides, simultaneously provide information in the same spectrum including both the peptide sequence and the structure of the N-linked glycan moiety. Selective screening and subsequent analysis of spectra of N-glycopeptides from large LC-MALDI-MS/MS datasets is supported by dedicated features implemented in Bruker ́s latest software tools. Application Note # ET-17 / MT-99 Characterization of the N-glycosylation Pattern of Antibodies by ESI – and MALDI Mass Spectrometry Introduction 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 detailed analysis of the N-glycosylation pattern present on antibodies is challenging due to the heterogeneous structure of this posttranslational modification. The glycan structure is highly dependent on the process by which the 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 antibody samples by utilizing two complementary MS based analysis techniques: Fast top-down analysis based on LC-MS accurate mass measurement of the intact antibody and the released heavy chain, performed on the maXis; a next generation ESI-ultra high-resolution (UHR)-TOF instrument. Comprehensive bottom-up characterization of the N-glycosylation patterns of various antibodies by nanoLCMALDI-TOF/TOF analysis of N-glycopeptides obtained from enzymatically digested antibodies; employing the ultrafleXtreme MALDI-TOF/TOF instrument. Experimental The analyses described here were performed on three different samples: a human IgG1 expressed in Chinese hamster ovary cells, a commercially available bovine IgG, and a sheep IgG from prefractionated sheep plasma. The human IgG was reduced and alkylated to release the glycosylated heavy chain (HC). An aliquot of the sample was separated by SDS-PAGE. The gel-separated heavy chain was then subjected to in-gel digestion using trypsin as an enzyme. The bovine IgG was digested in-solution after reduction and alkylation. The sheep IgG was part of a pre-fractionated plasma sample that had been reduced, alkylated using MMTS, and, after tryptic digestion, had been labelled with the iTRAQ4plex label (Applied Biosystems). The following experimental setup was used for the LC-ESI-UHR-TOF measurements on the intact glycoprotein: LC-ESI-UHR-TOF analysis of 5μg intact human IgG1 Fig. 1: The upper panel shows the raw data as acquired on the Bruker maXis. The lower panel shows the spectrum after Maximum Entropy deconvolution using the mass range colored in gray on the raw spectrum. Considering average atomic weights from organic sources according to Zhang et al., the theoretical mass of the mass labeled glycosylation form is 149081.79Da, which deviates 2ppm from the measured mass. 3043.5086 +MS, 7.5-8.8min #(87-102) 0 2 4 6 4 x10 Intens. 150