Computational protocol: iTRAQ-proteomics and bioinformatics analyses of mammary tissue from cows with clinical mastitis due to natural infection with Staphylococci aureus

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Protocol publication

[…] The cellular component (CC), molecular function (MF) and biological process (BP) of the proteins identified by iTRAQ were annotated by the Blast2GO software (http://www.blast2go.org/); GO functional classification and enrichment analysis were also performed to identify GO terms that were significantly enriched in differentially expressed proteins using DAVID analysis (http://david.abcc.ncifcrf.gov/).We used two different bioinformatic tools to examine the functional relationships among the differentially expressed proteins (DEP): the Dynamic Impact Approach (DIA) and the Ingenuity Pathway Analysis tool version 8.0 (IPA; Ingenuity® Systems, Inc, Redwood City, CA, http://www.ingenuity.com). Canonical pathway and regulatory network analyses were computed with IPA, using as a reference set the Ingenuity Pathways Knowledge Base. To identify the cellular mechanisms most related to mastitis, we searched the Ingenuity Pathways Knowledge Base for networks of interconnected modulated proteins, and for over-represented pathways. IPA is a software created to identify the most significant biological functions, canonical pathways, and networks embedded in a protein/gene set. The list was then uploaded into the IPA software that mapped and annotated 54 proteins, all involved in 3 main networks. IPA ranked networks depending on the reliability of the microarray results on the basis of prior published data. The statistical likelihood (Score) was used to rank the networks and ranged between 65 and 2 (Table  and Figure ).Figure 3To enhance the biological interpretation of the results, we used the DIA with the 55 differentially expressed proteins identified, considering in the analysis: KEGG pathways, each of the three main GO categories (BP, MF and CC) and, for predicting the presence of domains and important sites within proteins (INTERPRO).For DIA analysis, information from the freely-available online databases KEGG and DAVID (v6.7) was considered. A list of protein or gene identifiers (Entrez Gene IDs) from Bos taurus was uploaded all at once to extract and summarize functional annotations associated with groups of proteins or with each individual protein. Details of the DIA approach and its validation have been reported previously []. The direction of the impact (“flux”) was calculated according to our published procedure [], considering negative flux values as inhibited or downregulated and positive flux values as activated or upregulated. The impact value determines the biological significance of the change on a pathway and/or function by a treatment and/or change in physiological state. For this study, due to the abundance of significant GO terms and KEGG pathways from the DIA output, we considered as significant or more relevant from a biological standpoint those with calculated impact values above 30% of the total impact value of the top-impacted pathways. […]

Pipeline specifications

Software tools Blast2GO, DAVID, IPA
Application MS-based targeted proteomics
Organisms Bos taurus
Diseases Mastitis, Multiple Sclerosis