A method has been developed for release/isolation of O-glycans from glycoproteins in whole cell lysates for mass spectrometric analysis. Cells are lysed in SDS, which is then exchanged for urea and ammonium bicarbonate in a centrifugal filter, before treating with NH4OH to release O-glycans. Following centrifugation, O-glycans are recovered in the filtrate. Sonication achieves O-glycan release in 1 h. Combining the established protocol for filter-aided N-glycan separation, here optimized for enhanced PNGase F efficiency, with the developed O-glycan release method allows analysis of both N- and O-glycans from one sample, in the same filter unit, from 0.5 to 1 million cells. The method is compatible with subsequent analysis of the residual protein by liquid chromatography-mass spectrometry (LC-MS) after glycan release. The medium throughput approach is amenable to analysis of biological replicates, offering a simple way to assess the often subtle changes to glycan profiles accompanying differentiation and disease progression, in a statistically robust way.
A method has been developed for release/isolation of O-glycans from glycoproteins in whole cell lysates for mass spectrometric analysis. Cells are lysed in SDS, which is then exchanged for urea and ammonium bicarbonate in a centrifugal filter, before treating with NH4OH to release O-glycans. Following centrifugation, O-glycans are recovered in the filtrate. Sonication achieves O-glycan release in 1 h. Combining the established protocol for filter-aided N-glycan separation, here optimized for enhanced PNGase F efficiency, with the developed O-glycan release method allows analysis of both N- and O-glycans from one sample, in the same filter unit, from 0.5 to 1 million cells. The method is compatible with subsequent analysis of the residual protein by liquid chromatography–mass spectrometry (LC–MS) after glycan release. The medium throughput approach is amenable to analysis of biological replicates, offering a simple way to assess the often subtle changes to glycan profiles accompanying differentiation and disease progression, in a statistically robust way.
Glycoprotein glycosylation is altered in many diseases, including congenital disorders of glycosylation, as well as in cell differentiation. Alterations to the presence and location of glycan biosynthetic enzymes can manifest in subtle changes to glycan profiles. To determine which of these changes are statistically significant, analysis of multiple biological samples in parallel is needed. Protein glycosylation is also a major consideration in the pharmaceutical industry, with an increase in engineering of glycoprotein-based therapeutics. In therapeutic protein development, glycan structures can influence efficacy and stability. It is therefore important to understand glycan biosynthesis and to have a convenient means to assess the structures of glycans in order to further understand disease and genetic disorders linked to errors in glycosylation, as well as to potentially contribute to disease diagnostics and to inform development of biotherapeutics.
A filter-aid N-glycan separation (FANGS) method was recently developed, to release and isolate N-glycans, from a small number of culture whole cell lysates, for mass spectrometric analysis. This thesis presents a FANGS-based approach, to carry out O-glycan release, using β-elimination. Through method optimisation, O-glycan release can be achieved with 20 minutes of sonication, and gives comparable results to those from well-accepted overnight incubation methods. In addition, the thesis presents a streamlined protocol for a one-sample-one-pot approach to release N- and O-glycans from the same sample, in the same pot, with the potential to subsequently analyse protein remaining in the filter. Analysing both N- and O-glycans from one sample is important since in disease it is not always only one type of glycosylation that is altered. The developed method has been applied to mesenchymal stromal cells (MSCs), before and after differentiation into adipocytes, as well as genetically manipulated MSCs, in addition to undifferentiated and differentiated normal human urothelial cells. In both cell systems, changes could be observed in both the N- and O-glycan profiles. The approach offers insight into potential functional role of glycans in cellular processes such as differentiation and disease.
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