The filamentous fungus Trichoderma reesei is used for industrial production of secreted enzymes including carbohydrate active
enzymes, such as cellulases and hemicellulases. The production of many of these enzymes by T. reesei is influenced by the
carbon source it grows on, where the regulation system controlling hydrolase genes involves various signaling pathways. T.
reesei was cultivated in the presence of sorbitol, a carbon source that does not induce the production of cellulases and hemicellulases,
and then exposed to either sophorose or spent-grain extract, which are efficient inducers of the enzyme production. Specific
changes at phosphorylation sites were investigated in relation to the production of cellulases and hemicellulases using an
MS-based framework. Proteome-wide phosphorylation following carbon source exchange was investigated in the early stages of
induction: 0, 2, 5, and 10 min. The workflow involved sequential trypsin digestion, TiO2 enrichment, and MS analysis using
a Q Exactive mass spectrometer. We report on the identification and quantitation of 1721 phosphorylation sites. Investigation
of the data revealed a complex signaling network activated upon induction involving components related to light-mediated cellulase
induction, osmoregulation, and carbon sensing. Changes in protein phosphorylation were detected in the glycolytic pathway,
suggesting an inhibition of glucose catabolism at 10 min after the addition of sophorose and as early as 2 min after the addition
of spent-grain extract. Differential phosphorylation of factors related to carbon storage, intracellular trafficking, cytoskeleton,
and cellulase gene regulation were also observed.