title: An improved method for the detection and enrichment of low-abundant membrane and lipid raft-residing proteins.
abstract: A high degree of optimisation is required in native co-immunoprecipitation (co-IP) experiments with added challenges for low-abundant membrane proteins and masking by IgG molecules. Although in vivo tagged-protein purification avoids the IgG masking problem, modifying the terminus of the protein may result in conformational and post-translational modification changes. In this paper, we propose a method which combines four key aspects to improve the solubility and enrichment of low-abundant plasma membrane proteins using the urokinase plasminogen activator receptor (uPAR) as an example. As this GPI-linked receptor predominantly resides in lipid rafts (LR), we used a modified RIPA lysis buffer containing the non-ionic detergent, octyl-glucoside which solubilizes LRs to extract uPAR. This is followed by a modified crosslinking co-IP which covalently crosslinks the antibodies to the beads. Crosslinking allowed for a significant increase in the detection of uPAR with minimal IgG contamination using on-bead digestion or acid elution followed by digestion and analysis on high-throughput one-dimensional (nanoLC) MS/MS instrument (AbSciex 5600). To the best of our knowledge, this method of isolation is the first to be done to increase the yield of a low-abundant membrane protein and may be useful for the purification of other non-raft and raft-residing membrane proteins.
authors.affiliation: Department of Chemistry & Biomolecular Sciences, Faculty of Science, Macquarie University, NSW 2109, Australia.
authors.forename: Alison
authors.initials: A
authors.lastname: Kan
authors.forename: Abidali
authors.initials: A
authors.lastname: Mohamedali
authors.forename: Sock Hwee
authors.initials: SH
authors.lastname: Tan
authors.forename: Harish R
authors.initials: HR
authors.lastname: Cheruku
authors.forename: Iveta
authors.initials: I
authors.lastname: Slapetova
authors.forename: Ling Y
authors.initials: LY
authors.lastname: Lee
authors.forename: Mark S
authors.initials: MS
authors.lastname: Baker
mesh_terms: D005960:Glucosides; D047468:Immunoprecipitation / Q000379:methods*; D021962:Membrane Microdomains / Q000737:chemistry*; D008565:Membrane Proteins / Q000302:isolation & purification*; D055293:Receptors, Urokinase Plasminogen Activator / Q000302:isolation & purification; D053719:Tandem Mass Spectrometry
chemical_list: D005960:Glucosides; D008565:Membrane Proteins; D055293:Receptors, Urokinase Plasminogen Activator; C018619:octyl-beta-D-glucoside