TY - JOUR
T1 - Proteomic mapping of brain plasma membrane proteins
AU - Nielsen, Peter Aa
AU - Olsen, Jesper Velgaard
AU - Podtelejnikov, Alexandre V
AU - Andersen, Jens Roswalld
AU - Mann, Matthias
AU - Wisniewski, Jacek R
N1 - Keywords: Animals; Brain Chemistry; Cell Fractionation; Cell Membrane; Cerebral Cortex; Hippocampus; Membrane Proteins; Mice; Peptide Fragments; Peptide Mapping; Proteome; Proteomics; Sensitivity and Specificity
PY - 2005
Y1 - 2005
N2 - 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.
AB - 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.
U2 - 10.1074/mcp.T500002-MCP200
DO - 10.1074/mcp.T500002-MCP200
M3 - Journal article
C2 - 15684408
SN - 1535-9476
VL - 4
SP - 402
EP - 408
JO - Molecular and Cellular Proteomics
JF - Molecular and Cellular Proteomics
IS - 4
ER -