Research Article: Improved and semi-automated reductive β-elimination workflow for higher throughput protein O-glycosylation analysis

Date Published: January 17, 2019

Publisher: Public Library of Science

Author(s): Maximilianos Kotsias, Radoslaw P. Kozak, Richard A. Gardner, Manfred Wuhrer, Daniel I. R. Spencer, Frederique Lisacek.


Protein O-glycosylation has shown to be critical for a wide range of biological processes, resulting in an increased interest in studying the alterations in O-glycosylation patterns of biological samples as disease biomarkers as well as for patient stratification and personalized medicine. Given the complexity of O-glycans, often a large number of samples have to be analysed in order to obtain conclusive results. However, most of the O-glycan analysis work done so far has been performed using glycoanalytical technologies that would not be suitable for the analysis of large sample sets, mainly due to limitations in sample throughput and affordability of the methods. Here we report a largely automated system for O-glycan analysis. We adapted reductive β-elimination release of O-glycans to a 96-well plate system and transferred the protocol onto a liquid handling robot. The workflow includes O-glycan release, purification and derivatization through permethylation followed by MALDI-TOF-MS. The method has been validated according to the ICH Q2 (R1) guidelines for the validation of analytical procedures. The semi-automated reductive β-elimination system enabled for the characterization and relative quantitation of O-glycans from commercially available standards. Results of the semi-automated method were in good agreement with the conventional manual in-solution method while even outperforming it in terms of repeatability. Release of O-glycans for 96 samples was achieved within 2.5 hours, and the automated data acquisition on MALDI-TOF-MS took less than 1 minute per sample. This largely automated workflow for O-glycosylation analysis showed to produce rapid, accurate and reliable data, and has the potential to be applied for O-glycan characterization of biological samples, biopharmaceuticals as well as for biomarker discovery.

Partial Text

Glycosylation is one of the most common protein co-translational and post-translational modifications found in all domains of life.[1] There are two main types of protein glycosylation: N-glycosylation, where N-glycans are attached to asparagines in the tripeptide consensus sequence Asn-X-Ser/Thr (where X can be any amino acid except proline), and mucin-type-O-glycosylation where O-glycans are attached to the peptide chain through a Ser or Thr residue.[2]

We analysed the following samples: glycoprotein standards (BSM type I-S, fetuin, PSM type II) using the semi-automated method described. See supporting information (S1 File) section 2 and corresponding illustrations (Figures A and B and Tables A-C in S1 File) for details regarding O-glycosylation analysis of BSM type I-S, fetuin and PSM type II samples.

We have presented a largely automated O-glycosylation analysis workflow which was used for characterization and relative quantitation of O-glycans from commercially available standards (BSM type I-S, fetuin and PSM type II). Using fetuin (containing the O-glycans most commonly found in biopharmaceuticals)[37], BSM type I-S and PSM type II (containing complex and heterogeneous types of O-glycans) we demonstrate the efficacy and reliability of our system. To our knowledge, this is the first largely automated workflow for O-glycan release that has been adapted to a 96-well plate system to allow for its execution on a liquid handling robot, making this technology a good candidate for high throughput studies.




Leave a Reply

Your email address will not be published.