Proteomic screen for multiprotein complexes in synaptic plasma membrane from rat hippocampus by blue native gel electrophoresis and tandem mass spectrometry.
Neuronal synapses are specialized sites for information exchange between neurons. Many diseases, such as addiction and mood disorders, likely result from altered expression of synaptic proteins, or altered formation of synaptic complexes involved in neurotransmission or neuroplasticity. A detailed description of native multiprotein complexes in synaptic plasma membranes (PM) is therefore essential for understanding biological mechanisms and disease processes. For the first time in this study, two-dimensional Blue Native/SDS-PAGE electrophoresis, combined with tandem mass spectrometry, was used to screen multiprotein complexes in synaptic plasma membranes from rat hippocampus. As a result, 514 unique proteins were identified, of which 36% were integral membrane proteins. In addition, 19 potentially novel and known heterooligomeric multiprotein complexes were found, such as the SNARE and ATPase complexes. A potentially novel protein complex, involving syntaxin, synapsin I and Na+/K+ ATPase alpha-1, was further confirmed by co-immunoprecipitation and immunofluorescence staining. As demonstrated here, Blue Native-PAGE is a powerful tool for the separation of hydrophobic membrane proteins. The combination of Blue Native-PAGE and mass spectrometry could systematically identify multiprotein complexes.
Li
Xuanwen
X
Key Laboratory of Protein Chemistry and Developmental Biology of Education Committee, College of Life Sciences, Hunan Normal University, Changsha, 410081, PR China.
Xie
Chunliang
C
Jin
Qihui
Q
Liu
Mingjun
M
He
Quanyuan
Q
Cao
Rui
R
Lin
Yong
Y
Li
Jianglin
J
Li
Yan
Y
Chen
Ping
P
Liang
Songping
S
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D019295:Computational Biology / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006624:Hippocampus / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D008954:Models, Biological; D009474:Neurons / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D051381:Rats; D013347:Subcellular Fractions / Q000378:metabolism