Characterization of Degraded Proteins in Paintings Using Bottom-Up Proteomic Approaches: New Strategies for Protein Digestion and Analysis of Data

Chemical hydrolysis assisted by microwave irradiation has been proposed as an alternative method for the analysis of proteins in highly insoluble matrices. In this work, chemical hydrolysis was applied for the first time to detect degraded proteins from paintings and polychromies. To evaluate the performance of this approach, the number of identified peptides, protein sequence coverage (%), and PSMs were compared with those obtained using two trypsin-based proteomics procedures used for the analysis of samples from cultural heritage objects. It was found that chemical hydrolysis allowed the successful identification of all proteinaceous materials in all paint samples analyzed except for egg proteins in one extremely degraded sample. Moreover, in one sample, casein was only identified by chemical digestion. In general, chemical hydrolysis identified more peptides, more PSM’s, and greater sequence coverage in the samples containing caseins, and often also in animal glue, highlighting the great potential of this approach for the rapid digestion and identification of insoluble and degraded proteins from the field of the cultural heritage

Quantitative Site-Specific Phosphoproteomics of <i>Trichoderma reesei</i> Signaling Pathways upon Induction of Hydrolytic Enzyme Production

The filamentous fungus <i>Trichoderma reesei</i> 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 <i>T. reesei</i> is influenced by the carbon source it grows on, where the regulation system controlling hydrolase genes involves various signaling pathways. <i>T. reesei</i> 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