Protein solubility is a key component in the development of successful glycomics and glycoproteomics- based methodologies. Owing to their unique physical properties, a range of anionic detergents such as sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC) or chaotropic agents such as urea are commonly employed to facilitate protein solubilisation and denaturation. However, many of these detergents can interfere with different aspects of the downstream mass spectrometry analyses, thus impeding the advancement of comprehensive and inter-lab reproducible glycome and glycoproteome analyses.
In this study, we explored the use of two main detergents (SDS and SDC), denaturation conditions (DTT, heat) and urea to solubilise and denature a range of glycoproteins and cell extracts to determine the combinatory effects of these agents and its impact on subsequent N- and O-glycan characterisation. In addition, the use of desalting procedures to purify the released glycans were also simultaneously evaluated using Dowex (AG50) cation exchange and Porous Graphitised Carbon chromatography purification prior to PGC-LC-ESI mass spectrometry glycomics analyses.
Preliminary results indicate distinct differences in the N- and O-glycome profiles in relation to their column retention and elution as a result of the use of these combinatory agents despite applying the same analysis settings. The use of different reducing and denaturing conditions as well as different desalting protocols were found to significantly influence the signal intensity, separation capacity and retention times in PGC-LC ESI MS glycomics analyses.
An in-depth understanding on the impact of these conditions on downstream analyses is required to further improve sample preparation steps and reproducibility across laboratories to deliver reliable insights into glycome function and interactions in biological systems.