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1.
Dis Markers ; 2019: 3609789, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31191748

RESUMO

Although histologically benign, adamantinomatous craniopharyngioma (AC) pediatric brain tumor is a locally aggressive disease that frequently determines symptoms and hormonal dysfunctions related to the mass effect on the surrounding structures. Another typical feature of this benign neoplasm is the presence of voluminous liquid cysts frequently associated with the solid component. Even if studies have been devoted to the proteomic characterization of the tumor intracystic fluid, poor explorations have been performed on its solid part, principally investigated by transcriptomics technologies. In the present study, seven specimens of AC whole tumor tissue have been analyzed by LC-MS for a preliminary assessment of the proteomic profile by a top-down/bottom-up integrated approach. Thymosin beta 4, ubiquitin, calmodulin, S100 proteins, prothymosin α isoform 2, alpha-defensins 1-4, and fragments largely belonging to vimentin, hemoglobin, and glial fibrillary acidic protein characterized the intact proteome. The identification of alpha-defensins, formerly characterized in AC intracystic fluid, reinforces the hypothesis of a role for inflammation in tumor pathogenesis. A total number of 1798 unique elements were identified by a bottom-up approach with a special focus on the 433 proteins commonly characterized in the 85.7% of the samples analyzed. Their gene ontology classification evidenced the involvement of the adherence system, intermediate filaments, and actin cytoskeleton in tumor pathogenesis and of elements part of the Wnt, FGF, and EGFR signaling pathways. In addition, proteins involved in calcium modulation, innate immunity, inflammation, CCKR and integrin signaling, and gonadotropin-releasing hormone receptor pathways were also outlined. Further than confirming proteomic data previously obtained on AC intracystic fluid, these results offer a preliminary overview of the AC whole tissue protein phenotype, adding new hints towards the comprehension of this still obscure pediatric brain tumor.


Assuntos
Neoplasias Encefálicas/metabolismo , Craniofaringioma/metabolismo , Proteoma/metabolismo , Adolescente , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Encéfalo/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Criança , Craniofaringioma/genética , Craniofaringioma/patologia , Feminino , Humanos , Masculino , Proteoma/genética
2.
Artigo em Inglês | MEDLINE | ID: mdl-31202182

RESUMO

The multifactorial nature of Late Onset Alzheimer's Disease (LOAD), the AD form of major relevance on epidemiological and social aspects, has driven the original investigation by LC-MS and top-down proteomics approach of the protein repertoire of the brain tissue of TgCRND8 model mice fed with a diet deficient in B vitamins. The analysis of the acid-soluble fraction of brain tissue homogenates identified a list of proteins and peptides, proteoforms and PTMs. In order to disclose possible modulations, their relative quantification in wild type and AD model mice under both B vitamin deficient and control diets was performed. The levels of metallothionein III, guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 and brain acid soluble protein 1 showed statistically significant alterations depending on genotype, diet or both effects, respectively. Particularly, metallothionein III exhibited increased levels in TgCRND8 mice under B vitamin deficient diet with respect to wild type mice under both diets. Brain acid soluble protein 1 showed the opposite, revealing decreased levels in all diet groups of AD model mice with respect to wild type mice in control diet. Lower levels of brain acid soluble protein 1 were also observed in wild type mice under deficiency of B vitamins. These results, besides contributing to increase the knowledge of AD at molecular level, give new suggestions for deeply investigating metallothionein III and brain acid soluble protein 1 in AD.


Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Hiper-Homocisteinemia/metabolismo , Proteoma/metabolismo , Complexo Vitamínico B/análise , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Química Encefálica , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Cromatografia Líquida , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Hiper-Homocisteinemia/etiologia , Hiper-Homocisteinemia/genética , Masculino , Espectrometria de Massas , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteoma/química , Proteoma/genética , Complexo Vitamínico B/metabolismo
3.
Electrophoresis ; 37(7-8): 1015-26, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26719138

RESUMO

The lipoaspirate fluid (LAF) is emerging as a potentially valuable source in regenerative medicine. In particular, our group recently demonstrated that it is able to exert osteoinductive properties in vitro. This original observation stimulated the investigation of the proteomic component of LAF, by means of LC-ESI-LTQ-Orbitrap-MS top-down/bottom-up integrated approach, which represents the object of the present study. Top-down analyses required the optimization of sample pretreatment procedures to enable the correct investigation of the intact proteome. Bottom-up analyses have been directly applied to untreated samples after monodimensional SDS-PAGE separation. The analysis of the acid-soluble fraction of LAF by top-down approach allowed demonstrating the presence of albumin and hemoglobin fragments (i.e. VV- and LVV-hemorphin-7), thymosins ß4 and ß10 peptides, ubiquitin and acyl-CoA binding protein; adipogenesis regulatory factor, perilipin-1 fragments, and S100A6, along with their PTMs. Part of the bottom-up proteomic profile was reproducibly found in both tested samples. The bottom-up approach allowed demonstrating the presence of proteins, listed among the components of adipose tissue and/or comprised within the ASCs intracellular content and secreted proteome. Our data provide a first glance on the LAF molecular profile, which is consistent with its tissue environment. LAF appeared to contain bioactive proteins, peptides and paracrine factors, suggesting its potential translational exploitation.


Assuntos
Tecido Adiposo/química , Líquidos Corporais/química , Lipectomia , Proteoma/análise , Medicina Regenerativa , Tecido Adiposo/citologia , Líquidos Corporais/citologia , Cromatografia Líquida/métodos , Feminino , Humanos , Espectrometria de Massas/métodos , Proteoma/química , Proteoma/classificação , Proteômica/métodos
4.
EuPA Open Proteom ; 12: 13-21, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29900115

RESUMO

The proteome of the DAOY medulloblastoma cell line has been investigated by an LC-MS top-down platform. This approach, unlike bottom-up ones, allows identifying proteins and peptides in their intact/native forms, disclosing post-translational modifications, proteoforms and naturally occurring peptides. Indeed, 25 out of the 53 proteins identified, were not previously characterized in DAOY cells. Most of them were functionally interconnected, being mainly involved in binding, catalytic and structural activities, and metabolic processes. The top-down approach, applied in this preliminary study, disclosed the presence of several naturally occurring peptide fragments that characterize DAOY cells.

5.
Expert Opin Biol Ther ; 15 Suppl 1: S191-201, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26095945

RESUMO

OBJECTIVES: The aim of this study was to characterize ß and α thymosins and their proteoforms in various tissues and bodily fluids by mass spectrometry and to look at their association with a wide variety of pathologies. METHODS: A top-down proteomic platform based on high-performance liquid chromatography (HPLC) coupled to high-resolution LTQ-Orbitrap mass spectrometry (MS) was applied to the characterization of naturally occurring peptides. RESULTS: In addition to thymosin ß4 (Tß4) and ß10 (Tß10), several post-translational modifications of both these peptides were identified not only in bodily fluids but also in normal and pathological tissues of different origins. The analysis of tissue specimens allowed the characterization of different C-terminal truncated forms of Tß4 and Tß10 together with other proteolytic fragments. The sulfoxide derivative of both Tß4 and Tß10 and the acetylated derivatives at lysine residues of Tß4 were also characterized. Different proteoforms of prothymosin α, parathymosin α, thymosin α1 and thymosin α11 together with diverse proteolytic fragments were identified too. CONCLUSION: The clinical and prognostic significance and the origin of these proteoforms have to be deeply investigated.


Assuntos
Espectrometria de Massas/métodos , Timosina/análise , Adulto , Líquidos Corporais/química , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Recém-Nascido , Prognóstico , Precursores de Proteínas/análise , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Timalfasina , Timosina/análogos & derivados , Timosina/química , Timosina/metabolismo
6.
Mol Biosyst ; 11(6): 1668-83, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25909245

RESUMO

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 ß- 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.


Assuntos
Astrocitoma/química , Neoplasias Encefálicas/química , Meduloblastoma/química , Proteoma/análise , Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Criança , Pré-Escolar , Humanos , Meduloblastoma/metabolismo , Proteínas/análise , Proteínas/química , Proteínas/metabolismo , Proteoma/química , Proteoma/metabolismo , Proteômica
7.
J Proteome Res ; 13(11): 4594-606, 2014 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-25254300

RESUMO

Liquid chromatography in coupling with high-resolution ESI-LTQ-Orbitrap mass spectrometry was applied for a proteomic study of pediatric pilocytic astrocytoma brain tumor intracystic fluid by an integrated top-down/bottom-up platform. Both of the proteomic strategies resulted complementary and support each other in contributing to a wide characterization of the protein and peptide content of the tumor fluid. Top-down approach allowed to identify several proteins and peptides involved in different biological activities together with the characterization of interesting proteoforms such as fibrinopeptide A and its truncated form, fibrinopeptide B, complement C3f fragments, ß-thymosin peptides, ubiquitin, several apolipoproteins belonging to A and C families, apolipoprotein J and D, and cystatin C. Of particular interest resulted the identification of a N-terminal truncated cystatin C proteoform, likely involved in immune response mechanism modulations and the identification of oxidized and glycosylated apolipoproteins including disulfide bridge dimeric forms. The bottom-up approach confirmed some of the experimental data findings together with adding the characterization of high-molecular-mass proteins in the samples. These data could contribute to elucidate the molecular mechanisms involved in onset and progression of the disease and cyst development.


Assuntos
Astrocitoma/metabolismo , Líquido Cístico/metabolismo , Proteômica/métodos , Criança , Cromatografia Líquida de Alta Pressão , Cistatina C/metabolismo , Humanos , Espectrometria de Massas
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