Relative and absolute quantification of postsynaptic density proteome isolated from rat forebrain and cerebellum.
The postsynaptic density (PSD) of central excitatory synapses is essential for postsynaptic signaling, and its components are heterogeneous among different neuronal subtypes and brain structures. Here we report large scale relative and absolute quantification of proteins in PSDs purified from adult rat forebrain and cerebellum. PSD protein profiles were determined using the cleavable ICAT strategy and LC-MS/MS. A total of 296 proteins were identified and quantified with 43 proteins exhibiting statistically significant abundance change between forebrain and cerebellum, indicating marked molecular heterogeneity of PSDs between different brain regions. Moreover we utilized absolute quantification strategy, in which synthetic isotope-labeled peptides were used as internal standards, to measure the molar abundance of 32 key PSD proteins in forebrain and cerebellum. These data confirm the abundance of calcium/calmodulin-dependent protein kinase II and PSD-95 and reveal unexpected stoichiometric ratios between glutamate receptors, scaffold proteins, and signaling molecules in the PSD. Our data also demonstrate that the absolute quantification method is well suited for targeted quantitative proteomic analysis. Overall this study delineates a crucial molecular difference between forebrain and cerebellar PSDs and provides a quantitative framework for measuring the molecular stoichiometry of the PSD.
Cheng
Dongmei
D
Department of Human Genetics, Center for Neurodegenerative Disease, Emory University, Atlanta, Georgia 30322, USA.
Hoogenraad
Casper C
CC
Rush
John
J
Ramm
Elizabeth
E
Schlager
Max A
MA
Duong
Duc M
DM
Xu
Ping
P
Wijayawardana
Sameera R
SR
Hanfelt
John
J
Nakagawa
Terunaga
T
Sheng
Morgan
M
Peng
Junmin
J
D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000032:analysis; D002531:Cerebellum / Q000737:chemistry* / Q000166:cytology; D002853:Chromatography, Liquid; D000076122:Disks Large Homolog 4 Protein; D015180:Electrophoresis, Gel, Two-Dimensional; D020690:GTPase-Activating Proteins / Q000032:analysis; D047908:Intracellular Signaling Peptides and Proteins / Q000032:analysis; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000032:analysis; D009419:Nerve Tissue Proteins / Q000032:analysis*; D016548:Prosencephalon / Q000737:chemistry* / Q000166:cytology; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods; D051381:Rats; D017470:Receptors, Glutamate / Q000032:analysis; D013569:Synapses / Q000737:chemistry*
D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D020690:GTPase-Activating Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D020543:Proteome; D017470:Receptors, Glutamate; C112202:Syngap1 protein, rat; C033652:postsynaptic density proteins; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases