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

Alison Kan, Abidali Mohamedali, Sock Hwee Tan, Harish R. Cheruku, Iveta Slapetova, Ling Y. Lee, Mark S. Baker

Research output: Contribution to journalArticleResearchpeer-review

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.

LanguageEnglish
Pages299-304
Number of pages6
JournalJournal of Proteomics
Volume79
DOIs
Publication statusPublished - 1 Feb 2013

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Membrane Lipids
Urokinase Plasminogen Activator Receptors
Membrane Proteins
Immunoglobulin G
Immunoprecipitation
Crosslinking
Purification
Digestion
Proteins
Cell membranes
Post Translational Protein Processing
Detergents
Solubility
Blood Proteins
Buffers
Contamination
Throughput
Cell Membrane
Lipids
Molecules

Cite this

Kan, Alison ; Mohamedali, Abidali ; Tan, Sock Hwee ; Cheruku, Harish R. ; Slapetova, Iveta ; Lee, Ling Y. ; Baker, Mark S. / An improved method for the detection and enrichment of low-abundant membrane and lipid raft-residing proteins. In: Journal of Proteomics. 2013 ; Vol. 79. pp. 299-304.
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An improved method for the detection and enrichment of low-abundant membrane and lipid raft-residing proteins. / Kan, Alison; Mohamedali, Abidali; Tan, Sock Hwee; Cheruku, Harish R.; Slapetova, Iveta; Lee, Ling Y.; Baker, Mark S.

In: Journal of Proteomics, Vol. 79, 01.02.2013, p. 299-304.

Research output: Contribution to journalArticleResearchpeer-review

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