LC-MS/MS data was extracted using the extract_msn utility and searched against the UniProt Knowledgebase release 2010_11 using the Mascot search engine (version 2.3.01; Matrix Science) with tolerance for peptide mass

and fragment mass set to 15 ppm and 0.8 ppm, respectively. One missed trypsin cleavage and common variable modifications were accepted for peptide identification. After linear shift mass recalibration selleck chemicals llc the peptide mass window was narrowed to ± 5 ppm for final searches. The final search database contained all UniProtKB/Swiss-Prot entries for Mus musculus, Rattus norvegicus, and Homo sapiens including P00761, P00766, P02769, P11886, and P41921 as well as 22 UniProtKB/TrEMBL homologs to previously (in the course of this study) identified AMPAR complex constituents of these species. Proteins identified by only one specific MS/MS spectrum were not further considered. The average effective peptide FDR for all evaluated check details proteins (calculated as the number of corresponding peptides identified with a Mascot ion score ≥ 20 for the real database versus respective hits in a decoy database) was 0.029 (SD 0.021). Relative amino acid sequence coverage of proteins (Figures 1D and Table S2) was calculated as SC = Ni / (Ni + Nan), where Ni is the number of amino acid residues covered by identified peptides (Mascot e-value < 0.05, retrieval in > 2 independent APs) and Nan is the number of MS-accessible

(peptides within 740 < MW < 3,000 with trypsin cleavage C-terminal to the basic amino acids, but not N-terminal to proline; missed cleavages were not considered) but not Linifanib (ABT-869) identified amino acids in the respective database sequence. For peak volume-based quantification, m/z features along LC-MS scans were detected and quantified (as intensity × retention time × m/z width) using msInspect (Computational Proteomics Laboratory, Fred Hutchinson Cancer Research Center, Seattle,

WA, USA). After correction of m/z shifts (based on MS-sequenced peptides using an in-house written script), features were aligned between different LC-MS/MS runs and assigned to the peptides identified by Mascot (retention time tolerance: 3% or 1 min, m/z difference threshold: ± 5 ppm). The resulting peptide peak volumes (PVs) were used for two different quantification procedures. Protein Abundance Ratios (rPV). In AP versus control ( Figure 1), these were determined using the TopCorr method detailed in ( Bildl et al., 2012; Supplemental Experimental Procedures). Protein rPV values were plotted for each AB (AP versus controls, e.g., Figure 1B) to derive specificity thresholds from the resulting ratio distributions. Proteins were considered specifically copurified when rPV(versus IgG) > threshold(IgG) in both rat and mouse, and no cross-reactivity was indicated by rPV(versus KO) < threshold(versus KO) ( Figure 1). Relative Molar Abundances of Proteins.