title: Analysis of N-Glycosylation of Total Membrane Proteins.
abstract: 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.
authors.affiliation: Faculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovačića 1, 10 000, Zagreb, Croatia.
authors.forename: Tamara
authors.initials: T
authors.lastname: Pavić
authors.affiliation: Faculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovačića 1, 10 000, Zagreb, Croatia. ogornik@pharma.hr.
authors.forename: Olga
authors.initials: O
authors.lastname: Gornik
mesh_terms: D000818:Animals; D005591:Chemical Fractionation / Q000379:methods; D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006031:Glycosylation; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008565:Membrane Proteins / Q000032:analysis* / Q000302:isolation & purification; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000737:chemistry; D011134:Polysaccharides / Q000032:analysis* / Q000302:isolation & purification
chemical_list: D008565:Membrane Proteins; D011134:Polysaccharides; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40
keywords: Cloud point extraction; HILIC-UPLC; Membrane proteins; N-Glycans; Protein enrichment; Triton X-114