Pediatric Brain Tumors: Signatures from the Intact Proteome

Abstract

The present investigation aimed to explore the intact proteome of tissues of pediatric brain tumors of different WHO grades and localizations, including medulloblastoma, pilocytic astrocytoma, and glioblastoma, in comparison with the available data on ependymoma, to contribute to the understanding of the molecular mechanisms underlying the onset and progression of these pathologies. Tissues have been homogenized in acidic water–acetonitrile solutions containing proteases inhibitors and analyzed by LC–high resolution MS for proteomic characterization and label-free relative quantitation. Tandem MS spectra have been analyzed by either manual inspection or software elaboration, followed by experimental/theoretical MS fragmentation data comparison by bioinformatic tools. Statistically significant differences in protein/peptide levels between the different tumor histotypes have been evaluated by ANOVA test and Tukey’s post-hoc test, considering a p-value > 0.05 as significant. Together with intact protein and peptide chains, in the range of molecular mass of 1.3–22.8 kDa, several naturally occurring fragments from major proteins, peptides, and proteoforms have been also identified, some exhibiting proper biological activities. Protein and peptide sequencing allowed for the identification of different post-translational modifications, with acetylations, oxidations, citrullinations, deamidations, and C-terminal truncations being the most frequently characterized. C-terminal truncations, lacking from two to four amino acid residues, particularly characterizing the β-thymosin peptides and ubiquitin, showed a different modulation in the diverse tumors studied. With respect to the other tumors, medulloblastoma, the most frequent malignant brain tumor of the pediatric age, was characterized by higher levels of thymosin β4 and β10 peptides, the latter and its des-IS form particularly marking this histotype. The distribution pattern of the C-terminal truncated forms was also different in glioblastoma, particularly underlying gender differences, according to the definition of male and female glioblastoma as biologically distinct diseases. Glioblastoma was also distinguished for the peculiar identification of the truncated form of the α-hemoglobin chain, lacking the C-terminal arginine, and exhibiting oxygen-binding and vasoconstrictive properties different from the intact form. The proteomic characterization of the undigested proteome, following the top-down approach, was challenging to originally investigate the post-translational events that differently characterize pediatric brain tumors. This study provides a contribution to elucidate the molecular profiles of the solid tumors most frequently affecting the pediatric age, and which are characterized by different grades of aggressiveness and localization.

Citrullination Post-Translational Modification: State of the Art of Brain Tumor Investigations and Future Perspectives

“…In one review article, the perspectives of pan-PAD enzyme inhibitors and/or citrullinated protein signature applications in clinics for the early diagnosis and treatment of CNS diseases were also outlined. The different citrullinome profiles in male and female GBM patients revealed an interesting correlation with data from our recent top-down proteomic investigation of pediatric brain tumor tissues of different grades of aggressiveness and location, namely, medulloblastoma, pilocytic astrocytoma, ependymoma, and GBM [ 55 ]. Citrullination was identified in numerous peptide fragments of GFAP and vimentin naturally occurring in the intact proteome, and, in particular, in their C-terminal peptide fragments, which exhibited one or more PTM sites.…”

“…Vimentin and GFAP citrullination is produced by the PAD2 enzyme [ 23 , 35 ], and the identification of citrullinated peptides of these proteins in less aggressive histotypes of pediatric brain tumors would suggest a potential role in clinical prognostic applications. It is also noteworthy that citrullinated vimentin peptides with the same sequence as the citrullinated vimentin peptides found in pediatric brain tumor tissues [ 55 ] have been described for their immunoregulatory and anti-inflammatory properties in rheumatoid arthritis [ 57 ].…”