2.10. IPA functional enrichment analysis
Genes mapped from significantly upregulated or downregulated peptides and phosphopeptides were used to identify cellular and molecular processes, pathways and upstream regulators using Ingenuity pathway analysis (IPA) software (QIAGEN Redwood City,http://www.qiagen.com/ingenuity). Genes were queried against the Ingenuity knowledge database as the reference set. The regulated proteins, phosphoproteins and their log2-transformed SILAC ratios were uploaded into the IPA software, and the top canonical pathways associated with the uploaded phosphoproteins were listed along with the p values calculated using a right-tailed Fisher exact test. Upstream regulators refer to the upstream proteins that are responsible for causing changes in the phosphorylation and/or total expression levels of the queried genes/proteins in the dataset. Activation z-scores were calculated using IPA’s z-score algorithm to predict the overall activation or inhibition of the identified functional cellular processes/pathways and upstream regulators. A positive z-score (z-score > 0) implies an overall predicted activation of the process/pathway/upstream regulator, whereas a negative z-score (z-score < 0) implies an overall predicted inhibition or downregulation of the pathway/process/upstream regulator. Z-scores of ≥ 2 or ≤ −2 were considered by IPA to predict significant activation or inhibition, respectively. Cellular processes/upstream regulators with no z-scores imply that IPA was unable to generate prediction states for these functionalities.