Computational protocol: Proteomic-based identification of maternal proteins in mature mouse oocytes

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Protocol publication

[…] DTA files (Bioworks version 3.3) in ASCII format for each MS/MS spectrum with a minimum ion count of 8 were generated from the raw data for the peptide mass range of 400-8,000. The resulting spectra were independently searched against the International Protein Index Mouse database (ipi.MOUSE.v3.30, downloaded from http://ftp.ebi.ac.uk/pub/databases/IPI) containing 56450 entries by using SEQUEST analysis software (Bioworks version 3.3, ThermoFinnigan). Carbamidomethylation of cysteine was set as a fixed modification, and oxidized methionine was sought as a variable modification. The initial mass tolerances for protein identification on MS and MS/MS peaks were 10 ppm and 0.6 Da, respectively. Two missed cleavages were permitted. The criteria used for filtering peptides with low confidence scores were the following: cross-correlation values (Xcorr) greater than 2.0 and 2.5 were used for doubly charged ions and triply or higher charged ions, respectively; ΔCn values (difference in Xcorr with the next highest value) less than 0.1 were removed from the matched sequences. Singly charged ions were discarded because they were small in number. All output files were searched against the forward and reversed IPI mouse database separately, and FDR for all peptide-to-spectrum matches was calculated as FDR = # of False peptides/(# of True peptides + # of False peptides).For bioinformatics analysis, each IPI accession number was converted to an Entrez Gene ID according to the IPI protein cross-references file downloaded from http://ftp.ebi.ac.uk/pub/databases/IPI. We used Babelomics to find statistically over- and underrepresented GO categories in our oocyte proteome dataset. To compare the mouse oocyte proteome with other mouse tissue proteomes, we generated a combined database for the mouse based on the following mouse tissues and cell cultures characterized by LC-MS/MS: mouse heart [], liver [-], brain [,], lung [,], kidney [], spleen [], placenta[], cortical neurons cell culture[], sperm [], islet alpha-cell culture []. For enrichment analysis, our identified oocyte proteome was set as a test dataset and the combined mouse proteome was set as a reference. The enrichment analysis was done using 'fisher exact test', and all GO terms that were significant with adjusted P < 0.05 (after correcting for multiple term testing by using the FDR procedure of Bonferroni-Hochberg) were selected as overrepresented. An analysis of cellular processes influenced by the protein profile obtained was performed using PathwayStudio (v5.00) software (Ariadne Genomics, Inc., Rockville, MD). PathwayStudio includes an automated text-mining tool which enables the software to generate pathways from the PubMed database and other public sources. Each identified cellular process was confirmed through the PubMed/Medline hyperlink embedded in each node. The domain annotations were assigned using the Pfam database. […]

Pipeline specifications

Software tools Comet, Babelomics
Databases Gene IPI
Application MS-based untargeted proteomics
Organisms Mus musculus