engleski

Enrichment of hydrophobic membrane proteins using Triton X-114 and subsequent analysis of their N- glycosylation

Numerous proteins depend on correct glycosylation for their proper function and nearly all membrane, as well as secreted, proteins are glycosylated. Glycosylation of membrane proteins plays a crucial role in many processes including the intercellular recognition and intermolecular interactions on the cell surface. The composition of N-glycans attached to membrane proteins has not been sufficiently studied due to the lack of efficient and reproducible analytical methods. The aim of this study was to optimise cloud- point extraction (CPE) of membrane proteins with the non-ionic detergent Triton X- 114 and analyse their N-glycosylation using hydrophilic interaction liquid chromatography (HILIC-UPLC). Purification of isolated proteins from the excess of detergent proved to be the key step. Therefore, several purification procedures were tested to efficiently remove detergent, while retaining maximum protein recoveries. CPE showed to be an efficient method to simultaneously extract membrane and soluble proteins, which subsequently resulted in different N-glycan profiles of the aforementioned protein groups. The resulting protocol showed satisfactory reproducibility and potential for N-glycan analysis of both membrane and intracellular (soluble) proteins from different kinds of biological material. This method can be used as a new analytical tool for reliable detection and quantification of oligomannose and complex type N- glycans attached to membrane proteins, thus serving to distinguish between differences in cell types and states. The simple method was successfully optimised to generate reliable HILIC-UPLC profiles of N-glycans released from membrane proteins.

Cloud-point extraction (CPE) ; Membrane proteins ; N-glycosylation

S.I.: Glycans in personalised medicine.