Glycosylation affects structure, folding, and function of numerous proteins. Aberrant glycosylation has been shown to be associated with different diseases. Currently, there are several approaches for high throughput N- and O- linked glycan analysis of derivatised glycans such as ultra-performance liquid chromatography (UPLC), liquid chromatography mass spectrometry (LC-MS), capillary gel electrophoresis (CGE) 1. In our laboratory the routine method that has been found to be the best for glycan isomer separation and structural analysis is porous graphitised carbon liquid chromatography/mass spectrometry (PGC-LC-MS/MS) of reduced N- and O- glycans released form purified and complex mixtures of glycoproteins2. However the sample preparation is still carried out manually. Therefore there is a need for sensitive and robust high-throughput methods for the label-free release of glycans and their preparation for LC/MS analysis.
In our label-free method N-linked glycans are released using peptide N-glycosidase F (PNGaseF) and O-linked glycans subsequently released by reductive alkaline beta-elimination. The standard manual sample preparation for this glycan analysis is very labour-intensive, involving three days of bench work, and also the efficiency of glycan recovery could affect the reproducibility of the method during this workflow. To standardise and automate this process, we have successfully developed a high-throughput sequential N- and O-glycan release process using the liquid handling robot, Hamilton Nimbus (Bio-Strategy, Australia).
The Nimbus workflow accommodates a use of MPE2 (positive pressure module) device suited for high-throughput needs (96-well SPE plate) and Hamilton Heater Shaker (HHS). The results demonstrate that highly similar glycan profiles are measured when the Nimbus workflow is compared with the routine manual. The results were based on applying the workflow to analyse the N-linked glycans of a highly glycosylated mixture of immunoglobulins.
The development of this automated process for N- and O-glycan sample preparation from glycoproteins will enable reproducible, high throughput, sensitive analysis to be performed.