A Novel and Rapid Strategy for Labelling N-Glycans and Glycopeptides for Qualitative Analysis Using Uhplc-Esi-Ms

PS2 Poster session 2 Even numbers
Location (hall): 
Start/end time: 
Tuesday, July 2, 2019 - 15:45 to 17:15
Amez Martín

Manuela Amez Martín1,2, Concepcion Badia Tortosa1, Jenifer Hendel1, Richard Gardner1, Helene Ledru3, Carmen Galan3, Manfred Wuhrer2, Daniel Spencer1

1Ludger Ltd, Abingdon, United Kingdom, 2Leiden University Medical Centre, Centre for Proteomics and Metabolomics, Leiden, The Netherlands, 3School of Chemistry, University of Bristol, Cantock’s Close, Bristol BS8 1TS, Bristol, United Kingdom

Glycosylation can have significant effects on the clinical safety and efficacy of biopharmaceuticals [1,2]. As a result, both innovator drugs’ and biosimilars’ glycan profiles need to be fully characterized during the product lifecycle, from development to commercialization including batch-to-batch consistency and release to the market [2]. Additionally, a vast amount of research has been performed on the identification of disease biomarkers based on the glycosylation of biological samples [3,4,5]. Due to the high demand and institutional pressure for biopharmaceutical characterization [6] and the huge number of samples that a clinical setting is exposed to [7], there is a need for the development of a rapid, sensitive and selective method for glycan and glycopeptide analysis. Glycosylation analysis both at the N-glycan and glycopeptide level provide complementary information. N-glycan analysis gives specific information about the glycan structure and linkages by exoglycosidases analysis while glycopeptide analysis is a more favourable approach for the characterization of site-specific glycan compositions.

A new label for both N-glycan and glycopeptide analysis has been investigated. This label has an imidazolium group with a permanent positive charge that has been used as MS probe showing greater spectral peak intensities and lower limits of detection [8]. This new label synthesis is achieved in two steps. Glycoanalysis with this tag is quick and easy: it just requires 4 steps and no dry down steps are involved in the process, allowing it to be completed in as little as 2 hours. Final sample separation and identification is performed by tandem HILIC-UHPLC and ESI-MS. 

This poster will focus on the details of this new chemical derivatisation label. We will show that reliable data for both N-glycan and glycopeptide identification is generated using this rapid procedure with a small amount of glycoprotein (less than 10 μg for proteins with only one glycosylation site). The results obtained as a proof-of-concept for the analysis of antibodies and fetuin will be presented. As further optimization is required, the plans for future work will also be discussed.

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  2. Zhang, P.; Woen, S.; Wang, T.; Liau, B.; Zhao, S.; Chen, C.; Yang, Y.; Song, Z.; Wormald, M. R.; Yu, C.; et al. Challenges of Glycosylation Analysis and Control: An Integrated Approach to Producing Optimal and Consistent Therapeutic Drugs. Drug Discov. Today 2016, 21 (5), 740–765. https://doi.org/10.1016/j.drudis.2016.01.006.
  3. Kailemia, M. J.; Park, D.; Lebrilla, C. B. Glycans and Glycoproteins as Specific Biomarkers for Cancer. Analytical and Bioanalytical Chemistry. 2017, pp 395–410. https://doi.org/10.1007/s00216-016-9880-6.
  4. Costa, J.; Streich, L.; Pinto, S.; Pronto-Laborinho, A.; Nimtz, M.; Conradt, H. S.; de Carvalho, M. Exploring Cerebrospinal Fluid IgG N-Glycosylation as Potential Biomarker for Amyotrophic Lateral Sclerosis. Mol. Neurobiol. 2019. https://doi.org/10.1007/s12035-019-1482-9.
  5. Pavić, T.; Dilber, D.; Kifer, D.; Selak, N.; Keser, T.; Ljubičić, Đ.; Dugac, A. V.; Lauc, G.; Rumora, L.; Gornik, O. N-Glycosylation Patterns of Plasma Proteins and Immunoglobulin G in Chronic Obstructive Pulmonary Disease. J Transl Med 2018, 16 (323). https://doi.org/10.1186/s12967-018-1695-0.
  6. Fournier, J. A Review of Glycan Analysis Requirements. BioPharm Int. 2015, 28 (10).
  7. Walsh, G. Biopharmaceutical Benchmarks 2018; 2018; Vol. 36. https://doi.org/10.1038/nbt.4305.
  8. Galan, M. C.; Tran, A. T.; Bernard, C. Ionic-Liquid-Based Catch and Release Mass Spectroscopy Tags for Enzyme Monitoring. Chem. Commun. 2010, 46 (47), 8968–8970. https://doi.org/10.1039/c0cc04224b.