Proteomic mapping of brain plasma membrane proteins.
Proteomics is potentially a powerful technology for elucidating brain function and neurodegenerative diseases. So far, the brain proteome has generally been analyzed by two-dimensional gel electrophoresis, which usually leads to the complete absence of membrane proteins. We describe a proteomic approach for profiling of plasma membrane proteins from mouse brain. The procedure consists of a novel method for extraction and fractionation of membranes, on-membrane digestion, diagonal separation of peptides, and high-sensitivity analysis by advanced MS. Breaking with the classical plasma membrane fractionation approach, membranes are isolated without cell compartment isolation, by stepwise depletion of nonmembrane molecules from entire tissue homogenate by high-salt, carbonate, and urea washes followed by treatment of the membranes with sublytic concentrations of digitonin. Plasma membrane is further enriched by of density gradient fractionation and protein digested on-membrane by endoproteinase Lys-C. Released peptides are separated, fractions digested by trypsin, and analyzed by LC-MS/MS. In single experiments, the developed technology enabled identification of 862 proteins from 150 mg of mouse brain cortex. Further development and miniaturization allowed analysis of 15 mg of hippocampus, revealing 1,685 proteins. More that 60% of the identified proteins are membrane proteins, including several classes of ion channels and neurotransmitter receptors. Our work now allows in-depth study of brain membrane proteomes, such as of mouse models of neurological disease.
Nielsen
Peter Aa
PA
MDS Inc. Denmark, 5230 Odense M, Denmark.
Olsen
Jesper V
JV
Podtelejnikov
Alexandre V
AV
Andersen
Jens R
JR
Mann
Matthias
M
Wisniewski
Jacek R
JR
D000818:Animals; D001923:Brain Chemistry; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry*; D002540:Cerebral Cortex / Q000737:chemistry; D006624:Hippocampus / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification; D010449:Peptide Mapping; D020543:Proteome / Q000235:genetics / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*; D012680:Sensitivity and Specificity
D008565:Membrane Proteins; D010446:Peptide Fragments; D020543:Proteome