Computational protocol: Characterization of the functions and proteomes associated with membrane rafts in chicken sperm

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Protocol publication

[…] All MS and MS/MS raw spectra from each sample were searched using Proteome Discoverer v 1.4 (Thermo-Fisher Scientific) using a protein database for Gallus gallus downloaded from NCBI on March 15, 2016, with 46,000 entries. The peptide search was performed by two Sequest HT nodes, with the only difference being in static light dimethyl (+28.031Da) and heavy dimethyl (+36.076Da) of any lysine and peptide N-Terminus. The enzyme specificity was set to trypsin with two missed cleavages allowed. The peptide mass tolerance and fragment mass tolerance values were 10 ppm and 0.8 Da, respectively. A fixed carbamidomethyl modification of cysteine and variable methionine oxidation and deamidation of asparagine/glutamine were applied. Identified peptides were filtered for a maximum 1% false discovery rate (FDR) and minimum peptide confidence–high. Proteins were validated based on the presence of two or more unique peptides identified.For relative quantitation of heavy/light samples, the peak areas of detected precursor ions at each specific m/z corresponding to heavy and light peptides were generated from the precursor ion-based methyl-duplex algorithm in PD 1.4, with a mass tolerance at 4 ppm. […]

Pipeline specifications

Software tools Proteome Discoverer, Comet
Application MS-based untargeted proteomics
Organisms Gallus gallus, Homo sapiens
Diseases Cataract
Chemicals Biotin, Cholesterol, G(M1) Ganglioside, Octoxynol