Native proteomic analysis of protein complexes in murine intestinal brush border membranes.
Intestinal epithelial cell protrusions referred as microvilli or brush border membranes (BBMs) are specialized in the digestion, uptake, and transport of nutrients, trace elements and vitamins from intestinal lumen into the circulation. Disorders of intestinal absorption are common in human pathology and include serious defects such as malabsorption. A detailed description of native digestive protein complexes in BBMs is therefore essential for understanding the physiology and pathology of digestion and absorption. In this study, we employed blue native PAGE (BN-PAGE) technique to separate protein complexes from purified mouse intestinal BBMs. We found 23 distinct protein complexes, which were cut off from the gel, and their protein composition was determined by LC-MS/MS. A total of 55 individual proteins were identified including peptidases, enzymes of carbohydrate metabolism, membrane transporters, cytoskeletal proteins, chaperones, and regulatory enzymes. From the identified proteins, 50% represent molecules with at least one predicted transmembrane domain as predicted by SOSUI software. To the best of our knowledge, this work is the first attempt aimed to characterize the native membrane proteome of intestinal BBM. As demonstrated here, BN-PAGE is a powerful tool for the separation of not only mitochondrial, but also membrane hydrophobic proteins in general. In addition, BN-PAGE technique preserves metal-protein interactions, as shown by the presence of 65Zn in metalloprotein complexes, isolated from zinc-radiolabeled BBMs.
Babusiak
Marek
M
Institute of Hematology and Blood Transfusion, Prague, Czech Republic. babusiak@uhkt.cz
Man
Petr
P
Petrak
Jiri
J
Vyoral
Daniel
D
D000595:Amino Acid Sequence; D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D007413:Intestinal Mucosa / Q000378:metabolism*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008871:Microvilli / Q000378:metabolism*; D008969:Molecular Sequence Data; D020543:Proteome / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D015037:Zinc Radioisotopes / Q000737:chemistry
D020543:Proteome; D015037:Zinc Radioisotopes