Comparative analysis of chicken seminal plasma

Semen from 11 chickens was collected, supplemented with anti-proteases and centrifuged to separate sperm cells and fluids. 25µg of proteins for each chicken were included in-gel without fractionnation (1 band) followed by Coomassie Blue staining. After reduction and alkylation, proteins were in-gel digested with trypsin and peptide mixtures were analyzed by nanoLC-MS/MS using LTQ velos Orbitrap mass spectrometer. Raw data files were converted to MGF with Proteome Discoverer software (version 1.2; Thermo Fischer Scientific, San Jose, USA). Precursor mass range of 350-5000 Da and signal to noise ratio of 1.5 were the criteria used for generation of peak lists. In order to identify the proteins, the peptide and fragment masses obtained were matched automatically against the chordata section of a locally maintained copy of nr NCBI (19922528 sequences, download 08/21/2012). MS/MS ion searches were performed using MASCOT Daemon and search engine (version 2.3; Matrix Science, London, UK). The parameters used for database searches include trypsin as a protease with allowed two missed cleavage, carbamidomethylcysteine (+57 Da), oxidation of methionine (+16) and N-terminal protein acetylation (+42) as variable modifications. The tolerance of the ions was set to 5 ppm for parent and 0.8 Da for fragment ion matches