title: 15N-labeled brain enables quantification of proteome and phosphoproteome in cultured primary neurons.
abstract: Terminally differentiated primary cells represent a valuable in vitro model to study signaling events associated within a specific tissue. Quantitative proteomic methods using metabolic labeling in primary cells encounter labeling efficiency issues hindering the use of these cells. Here we developed a method to quantify the proteome and phosphoproteome of cultured neurons using (15)N-labeled brain tissue as an internal standard and applied this method to determine how an inhibitor of an excitatory neural transmitter receptor, phencyclidine (PCP), affects the global phosphoproteome of cortical neurons. We identified over 10,000 phosphopeptides and made accurate quantitative measurements of the neuronal phosphoproteome after neuronal inhibition. We show that short PCP treatments lead to changes in phosphorylation for 7% of neuronal phosphopeptides and that prolonged PCP treatment alters the total levels of several proteins essential for synaptic transmission and plasticity and leads to a massive reduction in the synaptic strength of inhibitory synapses. The results provide valuable insights into the dynamics of molecular networks implicated in PCP-mediated NMDA receptor inhibition and sensorimotor deficits.
authors: Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037, United States.
authors: Lujian
authors: L
authors: Liao
authors: Richard C
authors: RC
authors: Sando
authors: John B
authors: JB
authors: Farnum
authors: Peter W
authors: PW
authors: Vanderklish
authors: Anton
authors: A
authors: Maximov
authors: John R
authors: JR
authors: Yates
mesh_terms: D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D001923:Brain Chemistry; D002478:Cells, Cultured; D007553:Isotope Labeling / Q000379:methods; D013058:Mass Spectrometry; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D010622:Phencyclidine / Q000494:pharmacology; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D051381:Rats; D015203:Reproducibility of Results; D009435:Synaptic Transmission / Q000187:drug effects
chemical_list: D010750:Phosphoproteins; D020543:Proteome; D010622:Phencyclidine