Profiling of membrane proteins from human macrophages: comparison of two approaches.
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.
CNRS UMR 8576, and Université des Sciences et Techniques de Lille, Villeneuve d'Ascq, France.
Marie-Christine
MC
Slomianny
Annabelle
A
Dupont
Fatiha
F
Bouanou
Olivia
O
Beseme
Anne-Laure
AL
Guihot
Philippe
P
Amouyel
Jean-Claude
JC
Michalski
Florence
F
Pinet
D003902:Detergents / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006801:Humans; D039422:Integrin alpha3 / Q000378:metabolism; D007525:Isoelectric Focusing / Q000379:methods*; D037181:Lectins, C-Type / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism*; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry*; D017830:Octoxynol / Q000494:pharmacology; D040901:Proteomics / Q000379:methods*; D036141:Receptor, EphA7 / Q000737:chemistry; D011956:Receptors, Cell Surface / Q000378:metabolism; D015203:Reproducibility of Results; D012997:Solvents / Q000737:chemistry
D003902:Detergents; D039422:Integrin alpha3; D037181:Lectins, C-Type; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D012997:Solvents; D017830:Octoxynol; D036141:Receptor, EphA7