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Zeccola M.,Catholic University of the Sacred Heart | Longhi R.,CNR Institute of Chemistry of Molecular Recognition | Rossetti D.V.,Catholic University of the Sacred Heart | D'Angelo L.,Catholic University of the Sacred Heart | And 6 more authors.
Journal of Chromatography A | Year: 2012

A CE-tandem MS method was optimised and validated for selective and specific determination of LVV- and VV-hemorphin-7 peptides in cerebrospinal fluid. These two small peptides originate from haemoglobin beta chains. They possess relevant biological activity and recently a potential biomarker role in posterior cranial fossa paediatric brain tumour disease was evidenced. The separation was optimised using formic acid as background electrolyte and a water/methanol mixture, containing 0.1% (v/v) formic acid, as sheath liquid. The two peptides, differing in only one amino acid of the sequence at the N-terminal side were baseline separated in less than 15. min. The method allowed a very reduced and rapid sample pretreatment and was successfully applied to hemorphins determination in patient samples without matrix interferences. The method successfully passed bioanalytical validation showing linearity, accuracy and precision data on cerebrospinal fluid matrix within the acceptable values. The analysis of cerebrospinal fluid of patients affected by different posterior cranial fossa tumour forms confirmed our previous findings showing the absence of hemorphins in the pre-surgical cerebrospinal fluid and their presence in the post-ones and controls. The present method saves costs and time due to capillary electrophoresis miniaturisation and to the absence of chromatographic column and gradient elution and allows numerous injections per sample starting from few microlitres of cerebrospinal fluid. © 2012 Elsevier B.V. Source

Martelli C.,Catholic University of the Sacred Heart | Iavarone F.,Catholic University of the Sacred Heart | D'Angelo L.,Catholic University of the Sacred Heart | D'Angelo L.,Istituto Of Neurochirurgia Policlinico Universitario melli | And 16 more authors.
Molecular BioSystems | Year: 2015

A top-down/bottom-up integrated proteomic approach based on LC-MS and 2-DE analysis was applied for comparative characterization of medulloblastoma and pilocytic astrocytoma posterior cranial fossa pediatric brain tumor tissues. Although rare, primary brain tumors are the most frequent solid tumors in the pediatric age. Among them the medulloblastoma is the prevalent malignant tumor in childhood while pilocytic astrocytoma is the most common, rarely showing a malignant progression. Due to the limited availability of this kind of sample, the study was applied to pooled tumor tissues for a preliminary investigation. The results showed different proteomic profiles of the two tumors and evidenced interesting differential expression of several proteins and peptides. Top-down proteomics of acid-soluble fractions of brain tumor homogenates ascribed a potential biomarker role of malignancy to β- and α-thymosins and their truncated proteoforms and to C-terminal truncated (des-GG) ubiquitin, resulting exclusively detected or over-expressed in the highly malignant medulloblastoma. The bottom-up proteomics of the acid-soluble fraction identified several proteins, some of them in common with 2-DE analysis of acid-insoluble pellets. Peroxiredoxin-1, peptidyl-prolyl cis-trans isomerase A, triosephosphate isomerase, pyruvate kinase PKM, tubulin beta and alpha chains, heat shock protein HSP-90-beta and different histones characterized the medulloblastoma while the Ig kappa chain C region, serotransferrin, tubulin beta 2A chain and vimentin the pilocytic astrocytoma. The two proteomic strategies, with their pros and cons, well complemented each other in characterizing the proteome of brain tumor tissues and in disclosing potential disease biomarkers to be validated in a future study on individual samples of both tumor histotypes. © The Royal Society of Chemistry. Source

Desiderio C.,CNR Institute of Chemistry of Molecular Recognition | D'Angelo L.,Catholic University of the Sacred Heart | D'Angelo L.,Istituto Of Neurochirurgia Policlinico Universitario melli | Rossetti D.V.,Catholic University of the Sacred Heart | And 8 more authors.
Proteomics | Year: 2012

Posterior cranial fossa is the most frequent location of pediatric brain tumors. Its diagnosis is currently performed by postsurgery histopathology and the identification of biomarkers in cerebrospinal fluid (CSF) could provide a less invasive tool. Patient CSF was collected during surgery before the tumor removal (PRE-CSF) and 6 days after the resection (POST-CSF) and analyzed by top down LC-MS proteomics for comparison. The PRE-CSFs generally exhibited a less complex LC-MS profile than the relative POST-CSFs suggesting a suppressive role of the tumor toward proteins and peptides production or release. Particularly, a panel of peptides, identified as alpha- and beta-hemoglobin chains fragments, were generally absent in the PRE-CSF and present in the POST ones independently from contaminant blood hemoglobin. Among them, the LVV- and VV-hemorphin-7 showed the most repeatable trend and with a few remarkable exceptions: their unusual absence in POST surgery CSF was in fact interestingly correlated to the presence of tumor in the patient despite surgery due to metastases or to subtotal resection. These results ascribed a relevant biological role to LVV- and VV-h7 peptides in the disease and a strong potential as biomarkers. Their analysis in POST surgery CSF could be used to predict patient prognosis. © 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

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