Analysis of N-Glycosylation of Total Membrane Proteins
Glycosylation of membrane proteins plays a crucial role in various physiological events, including intercellular recognition and intermolecular interactions on the cell surface (Gornik et al., Biochim Biophys Acta 1820:1318–1326, 2012). To study composition and function of N-glycans on membrane proteins one has to have an efficient and reproducible analytical method, which includes protein extraction and analysis of glycans. In this chapter we provide an analytical approach that includes cloud-point extraction (CPE) of total membrane proteins with the non-ionic detergent Triton X-114 and subsequent analysis of their N-glycans using hydrophilic interaction liquid chromatography (HILIC)–UPLC/HPLC. The protocol presented here can be used for parallel analysis of both membrane and intracellular proteins.
Key wordsN-Glycans Membrane proteins Cloud point extraction Triton X-114 Protein enrichment HILIC-UPLC