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.