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

Abstract

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.

Structural and Quantitative Characterization of Mucin-Type O-Glycans and the Identification of O-Glycosylation Sites in Bovine Submaxillary Mucin

“…O-glycans in glycoproteins can alter the structure [26], function [4], and physicochemical properties of the protein to which they are attached [5], and BSM O-glycans have been characterized using various analytical methods. Permethylated BSM O-glycans have been identified by matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF) MS [27], which yielded different numbers of methylated groups [28], even though MALDI-TOF MS is not appropriate for analyzing O-glycans containing SA because these residues tend to be lost during ionization [29]. 1-phenyl-3-methyl-5-pyrazolone (PMP)-labeled O-glycans of BSM have been analyzed by LC-MS [30]; however, the separation of isomers and the sensitivity may be adversely affected when two PMP moieties are attached to one O-glycan [25], and detection based on ultraviolet absorption is less sensitive than fluorescence detection [21].…”

“…1-phenyl-3-methyl-5-pyrazolone (PMP)-labeled O-glycans of BSM have been analyzed by LC-MS [30]; however, the separation of isomers and the sensitivity may be adversely affected when two PMP moieties are attached to one O-glycan [25], and detection based on ultraviolet absorption is less sensitive than fluorescence detection [21]. Additionally, the average quantities of O-glycans of BSM have been identified through repeated experiments using MALDI-TOF MS [27].…”

Therapeutic Fc‐fusion proteins: Current analytical strategies

“…Indeed, in addition to N-glycans, Fc-fusion proteins often carry several O-glycosylation sites in the non-IgG domain. O-linked glycans require a different approach because they cannot be enzymatically cleaved using PNGase F. A common way to remove the O-glycans is via reductive β-elimination, after which, analysis can be performed with PGC-ESI-MS or MALDI-TOF [101,120,121]. This allows the challenging characterization of O-glycan structures, which in contrast to N-glycans, have highly diverse core structures [122].…”

Development of a 96-well plate sample preparation method for integratedN- andO-glycomics using porous graphitized carbon liquid chromatography-mass spectrometry

“…Currently, there are several high-throughput approaches for N-glycan analysis (mainly glycoprotein), [9][10][11] while less methods are available for O-glycomics. 12 Therefore, there is a high demand for high-throughput, reproducible and robust analytical methods for integrated N-and O-glycomics. Scaling up the conventional glycomics analysis to higher-throughput approaches especially with respect to the sample preparation workflow is of great importance for ensuring sufficient sample size providing more reliable results.…”