Aqueous polymer two-phase systems for the proteomic analysis of plasma membranes from minute brain samples.
Comprehensive knowledge about the plasma membrane protein profile of a given brain region, at defined developmental stages, will greatly foster the understanding of brain function and dysfunction. Protocols are required which selectively enrich plasma membranes from small brain regions, thereby resulting in high yields. Here, we present a suitable protocol that is based on aqueous polymer two-phase systems. It is material saving, easy to perform, fast, and low-priced. Evidence for its effectiveness was obtained by marker enzyme assays, immunoblot analyses, and mass spectrometry. Plasma membranes from all parts of the cells (somata, dendrites, and axons) were enriched, whereas there was a reduction of mitochondria and endoplasmic reticulum. The total of 15.0% of the initial activity of the plasma membrane marker was recovered, while the activity of the mitochondrial marker and the marker for the endoplasmic reticulum was 0.2% of the initial activity. Mass spectrometric analyses of proteins purified from approximately one-fourth of rat cerebellum (i.e., 80 mg of tissue) resulted in the identification of 525 different proteins, with 27.3% (gene ontology) or 38.2% (gene cards) being allocated to the plasma membrane. When accepting 4.7% of the initial mitochondrial marker activity and 2.9% of the initial activity of the marker for the endoplasmic reticulum as contaminations, the yield of the plasma membrane marker increased to 28.8%. Under these conditions, 586 different proteins were identified by mass spectrometry, 26.1-36.5% of which were plasma membrane proteins. Taken together, our protocol represents a powerful tool for the analysis of the plasma membrane subproteome of distinct brain regions.
Schindler
Jens
J
Animal Physiology Group, Department of Biology, University of Kaiserslautern, D- 67653 Kaiserslautern, Germany. jens.schindler@physiologie.uni-freiburg.de
Lewandrowski
Urs
U
Sickmann
Albert
A
Friauf
Eckhard
E
D000818:Animals; D001921:Brain / Q000648:ultrastructure*; D002462:Cell Membrane / Q000737:chemistry; D002531:Cerebellum / Q000737:chemistry*; D007425:Intracellular Membranes / Q000737:chemistry; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000032:analysis*; D044367:Phase Transition; D011108:Polymers; D040901:Proteomics; D051381:Rats; D012995:Solubility; D014867:Water
D008565:Membrane Proteins; D011108:Polymers; D014867:Water