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Profiling of membrane proteins from human macrophages: Comparison of two approaches
Author(s) -
Slomianny MarieChristine,
Dupont Annabelle,
Bouanou Fatiha,
Beseme Olivia,
Guihot AnneLaure,
Amouyel Philippe,
Michalski JeanClaude,
Pinet Florence
Publication year - 2006
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200500546
Subject(s) - membrane protein , integral membrane protein , peripheral membrane protein , receptor , membrane , chemistry , cell surface receptor , biochemistry , mannose receptor , proteomics , protein purification , biology , microbiology and biotechnology , macrophage , in vitro , gene
Abstract Macrophages are involved in various important biological processes and their functions are tightly regulated. Hydrophobic proteins are difficult to analyse by 2‐DE because of their intrinsic tendency to self‐aggregate during the first dimension (IEF). We have compared two protocols for extracting, separating and identifying membrane proteins from human macrophages by MALDI‐TOF MS. The first protocol used protein extraction by solvent, followed by 2‐DE and allowed us to identify 10% membrane proteins among the proteins identified a being like the peroxisome‐activated receptor delta. The second method is based on solubilizing the membranes with Triton X‐100, separating the proteins by anion‐exchange chromatography followed by SDS‐PAGE. This method allowed us to identify 49 membrane proteins, including four integral membrane proteins, ten type I, two type II and one type III membrane proteins. Several receptors were identified, including integrin alpha‐3 and ephrin type A receptor 7. Interestingly, several proteins involved in macrophage functions were identified, such as integrin alpha‐X and macrophage mannose receptor. These findings show that techniques are available to identify membrane proteins, but that they require large quantities of cells which means that they are not suitable for the limiting amounts of precious samples available from clinical studies.