An improved method for the detection and enrichment of low-abundant membrane and lipid raft-residing proteins

作者：Kan Alison; Mohamedali Abidali; Tan Sock Hwee; Cheruku Harish R; Slapetova Iveta; Lee Ling Y; Baker Mark S^*

来源：Journal of Proteomics, 2013, 79: 299-304.

DOI：10.1016/j.jprot.2012.11.019

• Crosslink co-immunoprecipitation (co-IP)
• Octyl-glucoside (OG)
• Lipid rafts (LRs)
• Urokinase plasminogen activator receptor (uPAR)

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.

• 出版日期2013-2-21