Caratterizzazione proteomica di fluidi e tessuti in diverse condizioni fisio-patologiche

This thesis has been focused on the proteomic characterization of human saliva from donors of different ages, starting from birth up to adult age, and pediatric brain tumor tissues. The first study has been performed in order to compare the acid-insoluble fraction of saliva from preterm with at-term newborns and adults and establish if differences exist. In the second study medulloblastoma and pilocytic astrocytoma pediatric brain tumor extracts have been compared. In both studies 2- DE analysis was coupled with high resolution tandem mass spectrometry (MS/MS). The proteomic characterization of the acid-insoluble fractions of saliva from preterm newborns allowed to integrate data previously obtained on the acid-soluble fraction by HPLC-electrospray ionization (ESI)-mass spectrometry (MS), and to evidence several differences between preterm newborns, at-term newborns and adults. Spots differentially expressed between the three groups, according to image analysis of the gels, were submitted to in-gel tryptic digestion and the peptide mixture analyzed by high performance HPLC-ESI-MS/MS for their characterization. By this strategy, we identified three over-expressed proteins in atterm newborns with respect to preterm newborns and adults (BPI fold-containing family A member 1, two proteoforms of annexin A1, and keratin type 1 cytoskeletal 13), and several over-expressed proteins in adults (fatty acid-binding protein, S100A6, S100A7, two proteoforms of S100A9, several proteoforms of prolactin-inducible protein, Ig kappa chain, two proteoforms of cystatin SN, one proteoform of cystatin S and several proteoforms of α-amylase 1). Moreover, for the first time, it was possible to assign by MS/MS four spots of human saliva 2-DE, already detected by other authors, to different proteoforms of S100A9. The strategy applied used a sequential staining protocol to the 2-DE gels, first with Pro-Q Diamond, that allows specific detection of phosphoproteins, and successively with total protein SYPRO Ruby stain. In the second study, proteomic analysis of two pediatric brain tumor tissues pointed out differences between medulloblastoma, the prevalent malignant tumor in childhood, and pilocytic astrocytoma, the most common, that only rarely shows a malignant progression. Due to the limited availability of bioptic tissue, the study was performed on pooled tumor tissues, and was focused on acid-insoluble fraction to integrate the characterization performed by a group of colleagues in Rome on the acid-soluble fraction by high performance HPLC-ESI-MS/MS. The results indicated that the two tumors exhibit different proteomic profiles and evidenced interesting differential expression of several proteins. Among them, peroxiredoxin- 1, peptidyl-prolyl cis–trans isomerase A, heterogeneous nuclear ribonucleoproteins A2/B1, mitochondrial isoform of malate dehydrogenase, nucleoside diphosphate kinase A, glutathione S-transferase P and fructose bisphosphate aldolase A resulted significantly over-expressed in medulloblastoma while glial fibrillary acidic protein, serotransferrin, α crystallin B chain, ferritin light chain, annexin A5, fatty acid-binding protein (brain), sorcin and apolipoprotein A-I resulted significantly over-expressed in pilocytic astrocytoma. In conclusion, the work done allowed to evidence the usefulness of using an integrated bottom-up/top-down approach, based on 2-DE-MS analysis and high performance MS in order to obtain a complete characterization of the proteome under investigation, revealing and identifying, not only peptides and small proteins, but also proteins with higher MW, that often it is not possible to identify by using exclusively a top-down ESI-MS approach.