Angel with the resolution and high 400th on a value of 60,000 at m / z Multiply charged up to five of the most intense ion scanning have collision-induced dissociation was fragmented in the linear ion trap. A dynamic exclusion window was applied AMPA Receptor in clinical trials in 60 s. All mass spectra were in tandem with standard collar ? lision energy of 35%, an isolation window 4 m / z and 1 collected microscan. Other Ger Teparameter including normal maximum injection time and automatic target acquisition with reinforcing GAIN of 500,000 and 250 ms ion embroidered, respectively for the Fourier transform mass spectrometry and 100 ms ion and 10,000, respectively, for MS LTQ / MS. The data were With the Xcalibur software and MS / MS peak lists centro Using the extracted msn were generated. exe executable file in the software-D built mon mascot.
The following parameters were set to create advanced lists : parent ions in the mass range 400 4500, ON-01910 no grouping of MS / MS scans, threshold 1000th Peak list was created for each frac tion ? analyzed and Mascot searches were performed for each fraction. The data were searched with Mascot server against sequences in Homo sapiens Swiss Prot TrEMBL database. This database contains Lt the UniProtKB / Swiss Prot protein knowledge Release-53. Integrated into a house with UniProtKB / TrEMBL Protein Database Release 36th A. Carbamidomethylation of cysteine was set as a fixed modification and oxidation of methionine as a variable modification for all MASCOT searches. Tolerances mass MS and MS / MS were set at 5 ppm and 0. There are 8, and the camera setting was specified as the case ESI.
Trypsin was called protease ? NAT, up to two missed cleavages were allowed. Shots proteins were validated automatically if it meets one of the criteria for ? ria: the identification of at least one peptide with a leading Mascot score of more than 50, at least the first two peptides, each with a rank score Mascot more than 35 years, or at least the first three ranking peptides each with a Mascot score greater than 30 as determined by the Mascot Search program, and using the automatic validation mod ule ? MFPaQ software developed in our house. Proteins Was identified with a single peptide best by manual inspection of MS / MS spectra CONFIRMS.
from all files validated results corresponding to fractions of a channel dimension gel MFPaQ was used to generate a single non-redundant list of proteins, by comparing groups of protein or proteins acc Access numbers and cre ating ? cluster groups of proteins found in different gel slices, if they have a common element. Comparisons were. Protein list on the comparison of groups of proteins, leading to different lists and software based MFPaQ these groups of proteins or divided as specific dependent Nts th ? whether they have common elements In order to assess the rate of false positives, all anf Nglichen base searches were performed using the Mascot decoy option, in other words, the data were searched on a combined database of sequences con ? real time specific protein and the corresponding protein returned is ? consequences. MFPaQ used the same criteria to validate decoy and defines the purpose of calculating the false discovery rate for each gel band ana lysed ?, and calculates the average FDR for all B Direction that. The same gel lane Episode
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