The resulting, uninterpreted MS/MS spectra were searched against the IPI mouse database 3.27 using Mascot algorithms to enable high-throughput BMN 673 solubility dmso protein identification. Wild-type and CSPα KO samples were subjected to 4-plex iTRAQ with technical replicates as described in Dávalos et al. (2010). The samples were trypsin digested,

labeled with iTRAQ tags, pooled, fractionated by cation exchange, and the individual peptides were run on an Applied Biosystems API Q-Star XL mass spectrometer. iTRAQ and protein identification were performed using the Paragon search algorithm (Shilov et al., 2007) in ProteinPilot 2.0 software against the IPI mouse database. Stringent criteria were used to identify potential CSPα clients. For the iTRAQ experiments at least two independent peptides with valid iTRAQ reporter ion ratios, exhibiting a minimum of two iTRAQ reporter ions with a summed S/N ratio > 9, were required to be included in the analysis. A cutoff was set at 0.7 for downregulated proteins and 1.4 for upregulated proteins or p < 0.01, whichever was more stringent was used for both DIGE and iTRAQ experiments. Principal components analysis was used to examine the consistency of the technical replicates and DIGE experiments, and proteins showing inconsistencies were disregarded. Proteins

that showed statistically significant changes by DIGE and iTRAQ were selected for protein level quantification using MRM methods. All analyses were carried out on a 5500 Q-TRAP instrument coupled to an online Waters nanoACQUITY Ultra High Pressure Liquid AZD9291 purchase Chromatography system. Data were initially processed using MRMPilot 2.0, Analyst 1.5 with MIDAS, and Multiquant 2.0 software.

Peptide identification was confirmed using MASCOT others 2.3. All raw mass spectrometry data are deposited in the Yale Protein Expression Database (YPED) (Shifman et al., 2007) and are publicly available through http://yped.med.yale.edu/repository (access code cqVUPu). Synaptosomal proteins, hippocampal neuronal culture extracts, or human tissue homogenates were subject to SDS-PAGE and western blotting. The proteins levels were quantified using conjugated IRDye secondary antibodies on a Li-COR Odyssey infrared imaging system. Actin and tubulin were used as internal controls. ATPase activity was assayed using colorimetric approach as described in Chamberlain and Burgoyne (1997a). Briefly, 1 μmol each of purified protein was incubated in ATPase assay buffer (10 mM MgCl2, 5 mM KCl, 50 mM Tris [pH 7.5]) for 5 min, followed by addition of 1 mM ATP to start the reaction. The free phosphate released was determined every 4 min using malachite green and measuring the absorbance at 650 nm. Hippocampal neuron cultures were immunostained using synaptophysin as presynaptic marker and MAP2 as a dendritic marker. Regions of interest were selected for each image such that areas with coalescing dendrites were excluded. Volocity 5.4.