Molecular constituents of the postsynaptic density fraction revealed by proteomic analysis using multidimensional liquid chromatography-tandem mass spectrometry.
Protein constituents of the postsynaptic density (PSD) fraction were analysed using an integrated liquid chromatography (LC)-based protein identification system, which was constructed by coupling microscale two-dimensional liquid chromatography (2DLC) with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) and an automated data analysis system. The PSD fraction prepared from rat forebrain was solubilized in 6 m guanidium hydrochloride, and the proteins were digested with trypsin after S-carbamoylmethylation under reducing conditions. The tryptic peptide mixture was then analysed with the 2DLC-MS/MS system in a data-dependent mode, and the resultant spectral data were automatically processed to search a genome sequence database for protein identification. In triplicate analyses, the system allowed assignments of 5264 peptides, which could finally be attributed to 492 proteins. The PSD contained various proteins involved in signalling transduction, including receptors, ion channel proteins, protein kinases and phosphatases, G-protein and related proteins, scaffold proteins, and adaptor proteins. Structural proteins, including membrane proteins involved in cell adhesion and cell-cell interaction, proteins involved in endocytosis, motor proteins, and cytoskeletal proteins were also abundant. These results provide basic data on a major protein set associated with the PSD and a basis for future functional studies of this important neural machinery.
Yoshimura
Yoshiyuki
Y
Department of Biochemistry, Faculty of Pharmaceutical Sciences, The University of Tokushima, Shomachi 1, Tokushima, Japan.
Yamauchi
Yoshio
Y
Shinkawa
Takashi
T
Taoka
Masato
M
Donai
Hitomi
H
Takahashi
Nobuhiro
N
Isobe
Toshiaki
T
Yamauchi
Takashi
T
D000818:Animals; D002452:Cell Count / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics*; D016548:Prosencephalon / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013569:Synapses / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013570:Synaptic Membranes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D008565:Membrane Proteins