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

Autor: Kotsias M; Ludger Ltd, Culham Science Centre, Abingdon, Oxfordshire, United Kingdom., Kozak RP; Ludger Ltd, Culham Science Centre, Abingdon, Oxfordshire, United Kingdom., Gardner RA; Ludger Ltd, Culham Science Centre, Abingdon, Oxfordshire, United Kingdom., Wuhrer M; Leiden University Medical Centre, Centre for Proteomics and Metabolomics, Leiden, Netherlands., Spencer DIR; Ludger Ltd, Culham Science Centre, Abingdon, Oxfordshire, United Kingdom.
Jazyk: angličtina
Zdroj: PloS one [PLoS One] 2019 Jan 17; Vol. 14 (1), pp. e0210759. Date of Electronic Publication: 2019 Jan 17 (Print Publication: 2019).
DOI: 10.1371/journal.pone.0210759
Abstrakt: 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.
Competing Interests: Ludger Ltd is a commercial bioscience company specialising in development and validation of glycoprofiling technology. The following authors declare competing financial interest(s): MK, RPK, RAG, DIRS are employed by Ludger Ltd. In addition, products from Ludger were used in this research. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
Databáze: MEDLINE
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