54 results on '"Rossetti DV"'
Search Results
2. Proteomic study of polocytic astrocytoma pediatric brain tumor intracystic fluid
- Author
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Inserra I., Iavarone F., Martelli C., D'Angelo L., Delfino D., Rossetti DV., Tamburrini G., Massimi L., Caldarelli M., Di Rocco C., Messana I., Castagnola M., and Desiderio C.
- Published
- 2014
3. N-acetyl cysteine directed detoxification of 2-hydroxyethyl methacrylate by adduct formation
- Author
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Claudia Desiderio, Vincenzo D'Antò, Diana Valeria Rossetti, Adriana Marquez Baquala, Rosa Valletta, Alessandro Lupi, Giuseppina Nocca, Gianrico Spagnuolo, Helmut Schweikl, Sandro Rengo, Nocca, G, D’Anto`, V, Desiderio, C, Rossetti, Dv, Valletta, Rosa, Baquala, Am, Schweikl, H, Lupi, A, Rengo, Sandro, Spagnuolo, G., G., Nocca, D'Antò, V., C., Desiderio, D. V., Rossetti, A. M., Baquala, H., Schweikl, A., Lupi, and Spagnuolo, Gianrico
- Subjects
chemistry/toxicity, Mice ,N-acetyl cysteine ,drug effects/metabolism, Mass Spectrometry, Methacrylate ,Cell Survival ,drug effects/metabolism, Fibroblast ,Intracellular Space ,Biophysics ,Bioengineering ,drug effects, Cell Survival ,Mass Spectrometry ,3T3 cells ,Adduct ,Biomaterials ,Mice ,medicine ,Extracellular ,Animals ,dental monomers ,High Pressure Liquid, Electrophoresi ,Cytotoxicity ,drug effects, Chromatography ,Cell damage ,Settore BIO/10 - BIOCHIMICA ,Chromatography, High Pressure Liquid ,Cell Death ,Chemistry ,Electrophoresis, Capillary ,HEMA ,3T3 Cells ,Capillary, Extracellular Space ,Fibroblasts ,medicine.disease ,In vitro ,Acetylcysteine ,medicine.anatomical_structure ,3T3 Cells, Acetylcysteine ,Biochemistry ,Mechanics of Materials ,Ceramics and Composites ,Methacrylates ,cytotoxicity ,Extracellular Space ,cytology/drug effects/metabolism, Intracellular Space ,chemistry/pharmacology, Animals, Cell Death ,Intracellular ,Cysteine - Abstract
Cytotoxicity of the dental resin monomer 2-hydroxyethyl methacrylate (HEMA) and the protective effects of N-acetyl cysteine (NAC) on monomer-induced cell damage are well demonstrated. The aim of our study was to analyze the hypothesis that the protection of NAC from HEMA cytotoxicity might be due to direct NAC adduct formation. To this end, using HPLC we first measured the actual intracellular HEMA concentrations able to cause toxic effects on 3T3-fibroblasts and then determined the decrease in intracellular and extracellular HEMA levels in the presence of NAC. In addition, by capillary electrophoresis coupled with mass spectrometry analysis (CE-MS), we evaluated NAC-HEMA adduct formation. HEMA reduced 3T3 cell vitality in a dose- and time-dependent manner. The concentration of HEMA inside the cells was 15-20 times lower than that added to the culture medium for cell treatment (0-8 mmol/L). In the presence of 10 mmol/L NAC, both intracellular and extracellular HEMA concentrations greatly decreased in conjunction with cytotoxicity. NAC-HEMA adducts were detected both in the presence and absence of cells. Our findings suggest that the in vitro detoxification ability of NAC against HEMA-induced cell damage occurs through NAC adduct formation. Moreover, we provide evidence that the actual intracellular concentration of HEMA able to cause cytotoxic effects is at least one magnitude lower than that applied extracellularly.
- Published
- 2010
4. Citrullination Post-Translational Modification: State of the Art of Brain Tumor Investigations and Future Perspectives.
- Author
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Rossetti DV, Muntiu A, Massimi L, Tamburrini G, and Desiderio C
- Abstract
The present review aims to describe the state of the art of research studies investigating the citrullination post-translational modification in adult and pediatric brain tumors. After an introduction to the deimination reaction and its occurrence in proteins and polypeptide chains, the role of the citrullination post-translational modification in physiological as well as pathological states, including cancer, is summarized, and the recent literature and review papers on the topic are examined. A separate section deals with the specific focus of investigation of the citrullination post-translational modification in relation to brain tumors, examining the state of the art of the literature that mainly concerns adult and pediatric glioblastoma and posterior fossa pediatric tumors. We examined the literature on this emerging field of research, and we apologize in advance for any possible omission. Although only a few studies inspecting citrullination in brain tumors are currently available, the results interestingly highlighted different profiles of the citrullinome associated with different histotypes. The data outlined the importance of this post-translational modification in modulating cancer invasion and chemoresistance, influencing key factors involved in apoptosis, cancer cell communication through extracellular vesicle release, autophagy, and gene expression processes, which suggests the prospect of taking citrullination as a target of cancer treatment or as a source of potential diagnostic and prognostic biomarkers for potential clinical applications in the future.
- Published
- 2023
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5. The Post-Translational Modifications of Human Salivary Peptides and Proteins Evidenced by Top-Down Platforms.
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Messana I, Manconi B, Cabras T, Boroumand M, Sanna MT, Iavarone F, Olianas A, Desiderio C, Rossetti DV, Vincenzoni F, Contini C, Guadalupi G, Fiorita A, Faa G, and Castagnola M
- Subjects
- Humans, Protein Processing, Post-Translational, Glycosylation, Proteolysis, Proteome, Salivary Proteins and Peptides
- Abstract
In this review, we extensively describe the main post-translational modifications that give rise to the multiple proteoforms characterized to date in the human salivary proteome and their potential role. Most of the data reported were obtained by our group in over twenty-five years of research carried out on human saliva mainly by applying a top-down strategy. In the beginning, we describe the products generated by proteolytic cleavages, which can occur before and after secretion. In this section, the most relevant families of salivary proteins are also described. Next, we report the current information concerning the human salivary phospho-proteome and the limited news available on sulfo-proteomes. Three sections are dedicated to the description of glycation and enzymatic glycosylation. Citrullination and N- and C-terminal post-translational modifications (PTMs) and miscellaneous other modifications are described in the last two sections. Results highlighting the variation in the level of some proteoforms in local or systemic pathologies are also reviewed throughout the sections of the manuscript to underline the impact and relevance of this information for the development of new diagnostic biomarkers useful in clinical practice.
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- 2023
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6. Pediatric Brain Tumors: Signatures from the Intact Proteome.
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Rossetti DV, Inserra I, Nesticò A, Vincenzoni F, Iavarone F, Messana I, Castagnola M, Massimi L, Tamburrini G, Caldarelli M, and Desiderio C
- Subjects
- Child, Female, Humans, Male, Peptides chemistry, Proteome metabolism, Proteomics methods, Brain Neoplasms metabolism, Cerebellar Neoplasms, Glioblastoma, Medulloblastoma
- 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.
- Published
- 2022
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7. Investigating Glioblastoma Multiforme Sub-Proteomes: A Computational Study of CUSA Fluid Proteomic Data.
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Moresi F, Rossetti DV, Vincenzoni F, Simboli GA, La Rocca G, Olivi A, Urbani A, Sabatino G, and Desiderio C
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- Brain Neoplasms genetics, Extracellular Vesicles metabolism, Glioblastoma genetics, Glycolysis, Humans, Proteome genetics, Brain Neoplasms metabolism, Glioblastoma metabolism, Proteome metabolism
- Abstract
Based on our previous proteomic study on Cavitating Ultrasound Aspirator (CUSA) fluid pools of Newly Diagnosed (ND) and Recurrent (R) glioblastomas (GBMs) of tumor core and periphery, as defined by 5-aminolevulinc acid (5-ALA) metabolite fluorescence, this work aims to apply a bioinformatic approach to investigate specifically into three sub-proteomes, i.e., Not Detected in Brain (NB), Cancer Related (CR) and Extracellular Vesicles (EVs) proteins following selected database classification. The study of these yet unexplored specific datasets aims to understand the high infiltration capability and relapse rate that characterizes this aggressive brain cancer. Out of the 587 proteins highly confidently identified in GBM CUSA pools, 53 proteins were classified as NB. Their gene ontology (GO) analysis showed the over-representation of blood coagulation and plasminogen activating cascade pathways, possibly compatible with Blood Brain Barrier damage in tumor disease and surgery bleeding. However, the NB group also included non-blood proteins and, specifically, histones correlated with oncogenesis. Concerning CR proteins, 159 proteins were found in the characterized GBM proteome. Their GO analysis highlighted the over-representation of many pathways, primarily glycolysis. Interestingly, while CR proteins were identified in ND-GBM exclusively in the tumor zones (fluorescence positive core and periphery zones) as predictable, conversely, in R-GBM they were unexpectedly characterized prevalently in the healthy zone (fluorescence negative tumor periphery). Relative to EVs protein classification, 60 proteins were found. EVs are over-released in tumor disease and are important in the transport of biological macromolecules. Furthermore, the presence of EVs in numerous body fluids makes them a possible low-invasive source of brain tumor biomarkers to be investigated. These results give new hints on the molecular features of GBM in trying to understand its aggressive behavior and open to more in-depth investigations to disclose potential disease biomarkers.
- Published
- 2022
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8. Adamantinomatous craniopharyngioma: advances in proteomic research.
- Author
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Desiderio C, Rossetti DV, Castagnola M, Massimi L, and Tamburrini G
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- Child, Cyst Fluid, Humans, Neoplasm Recurrence, Local, Proteomics, Craniopharyngioma, Pituitary Neoplasms
- Abstract
Background: Many efforts have been performed in the last decade to accomplish the genomic and proteomic characterization of pediatric adamantinomatous craniopharyngioma with the purpose to elucidate the molecular mechanisms underlying the onset and development of this pediatric brain tumor, its high recurrence rate, and, although classified as a histologically benign neoplasm, its aggressive behavior., Methods: The focus of this review is to perform the new comparison of the proteomic profiles of the solid component and the intracystic fluid of adamantinomatous craniopharyngioma based on our previous results, obtained by both the top-down and the bottom-up proteomic approaches, to disclose differences and similarities, and to discuss the results in the context of the most recent literature., Results and Conclusions: Proteins and peptides identified in the cyst fluid and in the solid component of adamantinomatous craniopharyngioma (AC) include beyond markers of inflammation (i.e., alpha-defensins), proteins involved in cell migration and protein degradation (i.e., beta-thymosin and ubiquitin peptides), whose main role might be in tumor growth and infiltration of the surrounding neural structures. These last appeared different in the solid components compared with the cyst fluid, missing their terminal part in the solid tissue, a feature generally associated to malignancies, which might represent a distinct molecular site for an aggressive behavior of AC.
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- 2021
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9. Glioblastoma CUSA Fluid Protein Profiling: A Comparative Investigation of the Core and Peripheral Tumor Zones.
- Author
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La Rocca G, Simboli GA, Vincenzoni F, Rossetti DV, Urbani A, Ius T, Della Pepa GM, Olivi A, Sabatino G, and Desiderio C
- Abstract
The present investigation aimed to characterize the protein profile of cavitating ultrasound aspirator fluid of newly diagnosed and recurrent glioblastoma comparing diverse zones of collection, i.e., tumor core and tumor periphery, with the aid of 5-aminolevulinic acid fluorescence. The samples were pooled and analyzed in triplicate by LC-MS following the shotgun proteomic approach. The identified proteins were then grouped to disclose elements exclusive and common to the tumor state or tumor zones and submitted to gene ontology classification and pathway overrepresentation analysis. The proteins common to the distinct zones were further investigated by relative quantitation, following a label free approach, to disclose possible differences of expression. Nine proteins, i.e., tubulin 2B chain, CD59, far upstream element-binding, CD44, histone H1.4, caldesmon, osteopontin, tropomyosin chain and metallothionein-2, marked the core of newly diagnosed glioblastoma with respect to tumor periphery. Considering the tumor zone, including the core and the fluorescence positive periphery, the serine glycine biosynthesis, pentose phosphate, 5-hydroxytryptamine degredation, de novo purine biosynthesis and huntington disease pathways resulted statistically significantly overrepresented with respect to the human genome of reference. The fluorescence negative zone shared several protein elements with the tumor zone, possibly indicating the presence of pathological aspects of glioblastoma rather than of normal brain parenchyma. On the other hand, its exclusive protein elements were considered to represent the healthy zone and, accordingly, exhibiting no pathways overrepresentation. On the contrary to newly diagnosed glioblastoma, pathway overrepresentation was recognized only in the healthy zone of recurrent glioblastoma. The TGFβ signaling pathway, exclusively classified in the fluorescence negative periphery in newly diagnosed glioblastoma, was instead the exclusive pathway classified in the tumor core of recurrent glioblastoma. These results, preliminary obtained on sample pools, demonstrated the potential of cavitron ultrasonic sur.
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- 2020
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10. Ependymoma Pediatric Brain Tumor Protein Fingerprinting by Integrated Mass Spectrometry Platforms: A Pilot Investigation.
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Rossetti DV, Massimi L, Martelli C, Vincenzoni F, Di Silvestre S, Scorpio G, Tamburrini G, Caldarelli M, Urbani A, and Desiderio C
- Abstract
Ependymoma pediatric brain tumor occurs at approximate frequencies of 10-15% in supratentorial and 20-30% in posterior fossa regions. These tumors have an almost selective response to surgery and relative and confirmed resistance to radiotherapy and chemotherapic agents, respectively. Alongside histopathological grading, clinical and treatment evaluation of ependymomas currently consider the tumor localization and the genomic outlined associated molecular subgroups, with the supratentorial and the posterior fossa ependymomas nowadays considered diverse diseases. On these grounds and in trying to better understand the molecular features of these tumors, the present investigation aimed to originally investigate the proteomic profile of pediatric ependymoma tissues of different grade and localization by mass spectrometry platforms to disclose potential distinct protein phenotypes. To this purpose, acid-soluble and acid-insoluble fractions of ependymoma tumor tissues homogenates were analyzed by LC-MS following both the top-down and the shotgun proteomic approaches, respectively, to either investigate the intact proteome or its digested form. The two approaches were complementary in profiling the ependymoma tumor tissues and showed distinguished profiles for supratentorial and posterior fossa ependymomas and for WHO II and III tumor grades. Top-down proteomic analysis revealed statistically significant higher levels of thymosin beta 4, 10 kDa heat shock protein, non-histone chromosomal protein HMG-17, and mono-/uncitrullinated forms ratio of the glial fibrillary acidic protein (GFAP) fragment 388-432 in supratentorial ependymomas-the same GFAP fragment as well as the hemoglobin alpha- and the beta-chain marked grade II with respect to grade III posterior fossa ependymomas. Gene ontology classification of shotgun data of the identified cancer and the non-cancer related proteins disclosed protein elements exclusively marking tumor localization and pathways that were selectively overrepresented. These results, although preliminary, seem consistent with different protein profiles of ependymomas of diverse grade of aggressiveness and brain region development and contributed to enlarging the molecular knowledge of this still enigmatic tumor.
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- 2020
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11. Exploring the brain tissue proteome of TgCRND8 Alzheimer's Disease model mice under B vitamin deficient diet induced hyperhomocysteinemia by LC-MS top-down platform.
- Author
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Delfino D, Rossetti DV, Martelli C, Inserra I, Vincenzoni F, Castagnola M, Urbani A, Scarpa S, Fuso A, Cavallaro RA, and Desiderio C
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- Alzheimer Disease genetics, Amyloid beta-Peptides metabolism, Animals, Brain Chemistry, Calmodulin-Binding Proteins genetics, Calmodulin-Binding Proteins metabolism, Chromatography, Liquid, Cytoskeletal Proteins genetics, Cytoskeletal Proteins metabolism, Disease Models, Animal, Female, Humans, Hyperhomocysteinemia etiology, Hyperhomocysteinemia genetics, Male, Mass Spectrometry, Metallothionein 3, Mice, Mice, Transgenic, Nerve Tissue Proteins genetics, Nerve Tissue Proteins metabolism, Proteome chemistry, Proteome genetics, Vitamin B Complex metabolism, Alzheimer Disease metabolism, Brain metabolism, Hyperhomocysteinemia metabolism, Proteome metabolism, Vitamin B Complex analysis
- Abstract
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., (Copyright © 2019. Published by Elsevier B.V.)
- Published
- 2019
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12. Investigating the Protein Signature of Adamantinomatous Craniopharyngioma Pediatric Brain Tumor Tissue: Towards the Comprehension of Its Aggressive Behavior.
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Martelli C, Serra R, Inserra I, Rossetti DV, Iavarone F, Vincenzoni F, Castagnola M, Urbani A, Tamburrini G, Caldarelli M, Massimi L, and Desiderio C
- Subjects
- Adolescent, Biomarkers, Tumor genetics, Biomarkers, Tumor metabolism, Brain metabolism, Brain Neoplasms genetics, Brain Neoplasms pathology, Child, Craniopharyngioma genetics, Craniopharyngioma pathology, Female, Humans, Male, Proteome genetics, Brain Neoplasms metabolism, Craniopharyngioma metabolism, Proteome metabolism
- Abstract
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.
- Published
- 2019
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13. Quantitative Determination of 18- β -Glycyrrhetinic Acid in HepG2 Cell Line by High Performance Liquid Chromatography Method.
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Nocca G, Callà C, Santini SA, Amalfitano A, Marigo L, Rossetti DV, Spagnuolo G, and Cordaro M
- Abstract
A reverse phase high performance liquid chromatographic (RP-HPLC) method was developed for identification and estimation of 18- β -glycyrrhetinic acid (GA) in HepG2 cell line. The analysis was carried out using a JASCO HPLC system with a C-18 (3 μ m) Supelco reversed phase column (150 x 4.7 mm) using a mobile phase of 80% CH
3 OH and 20% of CH3 CN: tetrahydrofuran: water (10:80:10, v/v/v). The method was linear in the concentration range of 1.5-120 μ g /mL (n = 5). The LOD and LOQ were determined based on standard deviation of the y-intercept and the slope of the calibration curve. The LOD and LOQ values were found to be 11.46 μ g/mL and 34.72 μ g/mL, respectively. The mean percentage recovery by standard addition experiments of GA is 92.4 % ± 5.2%. The intracellular GA concentration value, obtained as mean of five different determinations, was 45.8 ± 7.45 μ g/mL. We have developed a HPLC-UV method for quantitative determination of GA inside cells, with advantages in the cost reduction and economy of the analytical process.- Published
- 2018
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14. Integrated proteomic platforms for the comparative characterization of medulloblastoma and pilocytic astrocytoma pediatric brain tumors: a preliminary study.
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Martelli C, Iavarone F, D'Angelo L, Arba M, Vincenzoni F, Inserra I, Delfino D, Rossetti DV, Caretto M, Massimi L, Tamburrini G, Di Rocco C, Caldarelli M, Messana I, Castagnola M, Sanna MT, and Desiderio C
- Subjects
- Astrocytoma metabolism, Brain Neoplasms metabolism, Child, Child, Preschool, Humans, Medulloblastoma metabolism, Proteins analysis, Proteins chemistry, Proteins metabolism, Proteome chemistry, Proteome metabolism, Proteomics, Astrocytoma chemistry, Brain Neoplasms chemistry, Medulloblastoma chemistry, Proteome analysis
- Abstract
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.
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- 2015
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15. High-resolution mass spectrometry for thymosins detection and characterization.
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Cabras T, Iavarone F, Martelli C, Delfino D, Rossetti DV, Inserra I, Manconi B, Desiderio C, Messana I, Hannappel E, Faa G, and Castagnola M
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- Adult, Body Fluids chemistry, Chromatography, High Pressure Liquid methods, Humans, Infant, Newborn, Prognosis, Protein Precursors analysis, Protein Precursors chemistry, Protein Precursors metabolism, Protein Processing, Post-Translational, Proteomics methods, Thymalfasin, Thymosin analogs & derivatives, Thymosin chemistry, Thymosin metabolism, Mass Spectrometry methods, Thymosin analysis
- Abstract
Objectives: The aim of this study was to characterize β 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 β4 (Tβ4) and β10 (Tβ10), 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 Tβ4 and Tβ10 together with other proteolytic fragments. The sulfoxide derivative of both Tβ4 and Tβ10 and the acetylated derivatives at lysine residues of Tβ4 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.
- Published
- 2015
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16. Proteomic study of pilocytic astrocytoma pediatric brain tumor intracystic fluid.
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Inserra I, Iavarone F, Martelli C, D'Angelo L, Delfino D, Rossetti DV, Tamburrini G, Massimi L, Caldarelli M, Di Rocco C, Messana I, Castagnola M, and Desiderio C
- Subjects
- Child, Chromatography, High Pressure Liquid, Cystatin C metabolism, Humans, Mass Spectrometry, Astrocytoma metabolism, Cyst Fluid metabolism, Proteomics methods
- Abstract
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, β-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.
- Published
- 2014
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17. Proteomic characterization of pediatric craniopharyngioma intracystic fluid by LC-MS top-down/bottom-up integrated approaches.
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Martelli C, Iavarone F, Vincenzoni F, Rossetti DV, D'Angelo L, Tamburrini G, Caldarelli M, Di Rocco C, Messana I, Castagnola M, and Desiderio C
- Subjects
- Adolescent, Child, Child, Preschool, Chromatography, High Pressure Liquid, Female, Humans, Male, Mass Spectrometry, Peptide Fragments analysis, Trypsin, Craniopharyngioma chemistry, Cyst Fluid chemistry, Pituitary Neoplasms chemistry, Proteome analysis, Proteomics methods
- Abstract
The combination of top-down and bottom-up platforms was utilized for the LC-MS proteomic characterization of the intracystic fluid of adamantinomatous craniopharyngioma pediatric brain tumor disease. Proteins and peptides characterization was achieved by high-resolution LC-ESI-LTQ-Orbitrap-MS analysis while low-resolution LC-ESI-IT-MS was applied for the complete screening of the samples and the evaluation of the protein distribution within patients. Top-down analyses were applied to liquid/liquid extracted samples while bottom-up analyses were performed after trypsin digestion of both untreated and pretreated samples. The two proteomic approaches were complementary for the characterization of the proteome of craniopharyngioma intracystic fluid. Proteins and peptides involved in inflammation, mineralization processes and lipid transport were identified, in agreement with the calcium flecks, cholesterol granules and bone residues characteristic of this fluid. Apolipoprotein A-I, A-II, C-I and J, hemoglobin fragments, ubiquitin, α-2-HS-glycoprotein or fetuin A, α-1-antichymotrypsin, vitamin D binding protein, and α-1-acid glycoprotein were characterized. These data could be relevant for the comprehension of the processes involved in the pathogenesis of the disease and the development of the cyst and could contribute to the individuation of therapeutic targets for the reduction of the cyst volume delaying and/or avoiding invasive surgical treatments., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2014
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18. Quantitative analysis of thymosin β4 in whole saliva by capillary electrophoresis–mass spectrometry using multiple ions monitoring (CE-MIM-MS.).
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Rossetti DV, Martelli C, Longhi R, Iavarone F, Castagnola M, and Desiderio C
- Subjects
- Adult, Aged, Amino Acid Sequence, Female, Humans, Linear Models, Male, Middle Aged, Molecular Sequence Data, Reproducibility of Results, Sensitivity and Specificity, Electrophoresis, Capillary methods, Mass Spectrometry methods, Saliva chemistry, Thymosin analysis
- Abstract
Thymosin β4 (Tβ4) is a peptide present in almost any tissue and in extracellular media in mammals, having multiple amazing functions as wound healing, stimulation of angiogenesis, and suppression of inflammation. This study describes its determination in saliva through CE-MS using multiple ions monitoring scan mode by isolating the four most intense multicharged ions present in the MS spectra of the peptide. This scan modality, by reducing the baseline noise and interferences, increases the sensitivity and specificity in biological matrices. The CE-MS separation was optimized by studying different parameters influencing CE analysis, sample injection, and MS ionization, that is, the nebulizer gas flow, the sheath liquid, and BGE composition. The proposed technique can unambiguously identify in short time Tβ4 in saliva after a very fast and reduced sample pretreatment procedure. The method was validated for quantitation showing linearity of the response in the range 0.25 (lower limit of quantification) to 4 μM (average R2 0.996 ± 0.005) and intra- and interassay precision and accuracy at three different concentrations with RSD values in the range of 7–16%. It was successfully applied to the analysis of Tβ4 in whole saliva showing a variable peptide content from individual to individual (in the range of 0.3–1.4 μM) and in different days from the same individual. CE-MS in multiple ions monitoring scan mode provides a fast, selective, and economic method requiring only very few microliters of sample.
- Published
- 2013
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19. In vitro cellular detoxification of triethylene glycol dimethacrylate by adduct formation with N-acetylcysteine.
- Author
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Spagnuolo G, Desiderio C, Rivieccio V, Amato M, Rossetti DV, D'Antò V, Schweikl H, Lupi A, Rengo S, and Nocca G
- Subjects
- Acetylcysteine chemistry, Animals, Cell Culture Techniques, Cell Survival drug effects, Chromatography, High Pressure Liquid methods, Coloring Agents, Cytosol chemistry, Dose-Response Relationship, Drug, Drug Interactions, Electrophoresis, Capillary, Extracellular Space chemistry, Methacrylates analysis, Mice, Polyethylene Glycols analysis, Polyethylene Glycols chemistry, Polymethacrylic Acids analysis, Polymethacrylic Acids chemistry, Protective Agents chemistry, Swiss 3T3 Cells, Tandem Mass Spectrometry methods, Tetrazolium Salts, Thiazoles, Time Factors, Ultraviolet Rays, Acetylcysteine pharmacology, Fibroblasts drug effects, Polyethylene Glycols toxicity, Polymethacrylic Acids toxicity, Protective Agents pharmacology
- Abstract
Objective: Various protective effects of N-acetylcysteine (NAC) against triethylene glycol dimethacrylate (TEGDMA)-induced cell damage have been demonstrated, but so far there is no evidence on NAC direct monomer detoxification mechanism. Here, we hypothesized that NAC might reduce TEGDMA cytotoxicity due to direct NAC adduct formation., Methods: We measured the cytotoxic effects of TEGDMA in presence and in absence of NAC by MTT test. Then we analyzed the presence of TEGDMA-NAC adduct formation in extracellular and intracellular compartments by capillary electrophoresis-UV detection (CE-UV) and capillary electrophoresis-mass spectrometry (CE-MS) analytical techniques. Moreover, we quantified the effective intracellular and extracellular TEGDMA concentrations through HPLC in the presence and absence of 10 mmol/L NAC., Results: TEGDMA reduced 3T3 cell vitality in a dose- and time-dependent manner, while NAC decreased monomer cytotoxicity and extracellular monomer concentrations by a direct reaction with TEGDMA. The adducts between the two molecules were detected both in the presence and absence of cell. Moreover a signal ascribed to the methacrylic acid was present in the CE-UV electropherogram of cellular lysates obtained after incubation with TEGDMA., Significance: Our results suggest that in vitro detoxification capability of NAC against TEGDMA-induced cell damage might occur also through the formation of NAC-TEGDMA adduct., (Copyright © 2013 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)
- Published
- 2013
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20. Identification of thymosins β4 and β 10 in paediatric craniopharyngioma cystic fluid.
- Author
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Desiderio C, Martelli C, Rossetti DV, Di Rocco C, D'Angelo L, Caldarelli M, Tamburrini G, Iavarone F, Castagnola M, Messana I, Cabras T, and Faa G
- Subjects
- Adolescent, Child, Child, Preschool, Chromatography, High Pressure Liquid, Craniopharyngioma surgery, Endoscopy, Female, Humans, Male, Mass Spectrometry, Multiprotein Complexes, Pituitary Neoplasms surgery, Craniopharyngioma cerebrospinal fluid, Craniopharyngioma metabolism, Pituitary Neoplasms cerebrospinal fluid, Pituitary Neoplasms metabolism, Thymosin cerebrospinal fluid
- Abstract
Background: Adamantinomatous craniopharyngioma is the third most recurrent paediatric brain tumour. Although histologically benign, it behaves aggressively as a malignant tumour due to invasion of the hypothalamus and visual pathways. Surgery is still the first and almost the only mode of treatment, although serious damage can occur as a consequence of tumour localization. The proteomic characterization of the intracystic tumoural fluid could contribute to the comprehension of the tumorigenesis processes and to the development of therapeutic targets to reduce cyst volume, allowing less invasive surgery and/or delay of the radical resection of the tumour mass and the collateral serious effects., Methods: Intracystic fluid was analysed by a LC-ESI-IT-MS top-down platform after acidification, deproteinization and chloroform liquid/liquid extraction., Findings: Thymosin β4 and β10 peptides were for the first time identified in the intracystic fluid of adamantinomatous craniopharyngioma by low- and high-resolution MS analysis coupled with LC. The two peptides showed the same distribution trend in the analysed samples. Thymosin β4 and β10 were present in 77 % of the analysed samples. These peptides were not found in the cerebrospinal fluid available for two patients., Interpretation: The presence of β-thymosins in the intracystic fluid of the tumour confirmed the secretion of these proteins in the extracellular environment. Due to their G-actin-sequestering activity and antiapoptotic and anti-inflammatory properties, these peptides could be strictly involved in both tumour progression and cyst development and growth.
- Published
- 2013
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21. Development and validation of a capillary electrophoresis tandem mass spectrometry analytical method for the determination of Leu-Val-Val- and Val-Val-hemorphin-7 peptides in cerebrospinal fluid.
- Author
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Zeccola M, Longhi R, Rossetti DV, D'Angelo L, Tamburrini G, Di Rocco C, Giardina B, Castagnola M, and Desiderio C
- Subjects
- Brain Neoplasms diagnosis, Child, Child, Preschool, Female, Humans, Male, Brain Neoplasms cerebrospinal fluid, Electrophoresis, Capillary methods, Hemoglobins cerebrospinal fluid, Peptide Fragments cerebrospinal fluid, Tandem Mass Spectrometry methods
- Abstract
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., (Copyright © 2012 Elsevier B.V. All rights reserved.)
- Published
- 2012
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22. Cerebrospinal fluid top-down proteomics evidenced the potential biomarker role of LVV- and VV-hemorphin-7 in posterior cranial fossa pediatric brain tumors.
- Author
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Desiderio C, D'Angelo L, Rossetti DV, Iavarone F, Giardina B, Castagnola M, Massimi L, Tamburrini G, and Di Rocco C
- Subjects
- Adolescent, Amino Acid Sequence, Biomarkers, Tumor chemistry, Brain Neoplasms pathology, Child, Child, Preschool, Cranial Fossa, Posterior pathology, Female, Hemoglobins chemistry, Humans, Infant, Infant, Newborn, Male, Molecular Sequence Data, Peptide Fragments chemistry, Biomarkers, Tumor cerebrospinal fluid, Brain Neoplasms cerebrospinal fluid, Hemoglobins cerebrospinal fluid, Peptide Fragments cerebrospinal fluid, Proteomics methods
- Abstract
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.)
- Published
- 2012
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23. Identification of glutathione-methacrylates adducts in gingival fibroblasts and erythrocytes by HPLC-MS and capillary electrophoresis.
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Nocca G, Ragno R, Carbone V, Martorana GE, Rossetti DV, Gambarini G, Giardina B, and Lupi A
- Subjects
- Biocatalysis, Cells, Cultured, Cysteine metabolism, Cytoplasm metabolism, Erythrocytes enzymology, Ethacrynic Acid metabolism, Extracellular Space metabolism, Fibroblasts enzymology, Gingiva cytology, Gingiva enzymology, Glutathione analysis, Glutathione Transferase metabolism, Humans, Materials Testing, Methacrylates analysis, Molecular Conformation, Polyethylene Glycols analysis, Polyethylene Glycols metabolism, Polymethacrylic Acids analysis, Polymethacrylic Acids metabolism, Chromatography, High Pressure Liquid, Chromatography, Micellar Electrokinetic Capillary, Erythrocytes metabolism, Fibroblasts metabolism, Gingiva metabolism, Glutathione metabolism, Methacrylates metabolism, Tandem Mass Spectrometry
- Abstract
Objectives: Methacrylic monomers are released, from dental composite resins, either into the oral cavity or in pulpal tissues, where they can cause local or systemic adverse effects. The mechanisms of these effects are not well understood, probably because such molecules can act at different levels also inducing a depletion of intracellular glutathione (GSH). GSH can detoxify methacrylates by conjugating their α,β-unsaturated carbon-carbon moiety to the thiol group, with the catalysis of glutathione S-transferases (GST). This reaction determines a GSH cellular depletion and belongs to the metabolism of α,β-unsaturated esters, protecting the body against the toxic effects of electrophiles. On the basis of the above considerations, this work aim is to set up a method for the detection of the adducts formed by methacrylic monomers with GSH in cells using HPLC coupled to mass spectrometry (HPLC-MS) and micellar electrokinetic capillary chromatography (MECK) techniques., Methods and Results: Adducts of glutathione with triethylene glycol dimethacrylate (TEGDMA) and hydroxyethyl methacrylate (HEMA) were incontrovertibly identified by HPLC-MS and MECK in human gingival fibroblasts and erythrocytes, both outside and inside cells. Molecular docking simulations of HEMA and TEGDMA in the experimental structure of glutathione S-transferase, are also reported to rationalize the effectiveness of such enzyme in the catalysis of the above described reaction., Significance: The setup of a method for the identification of GSH-methacrylate adducts allows to determine when the metabolic pathway involving such compounds is employed by cells for the detoxification of monomers leached from composite resins., (Copyright © 2011 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)
- Published
- 2011
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24. Capillary electrophoresis--mass spectrometry: recent trends in clinical proteomics.
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Desiderio C, Rossetti DV, Iavarone F, Messana I, and Castagnola M
- Subjects
- Animals, Biomarkers analysis, Electrophoresis, Capillary trends, Humans, Kidney Diseases blood, Kidney Diseases diagnosis, Kidney Diseases urine, Mass Spectrometry trends, Proteomics trends
- Abstract
The increasing attention now paid to the elucidation of human proteome strengthened the development of analytical instruments able to provide reliable proteins and peptides quantitation and characterization in biological fluids and tissues. Emerging from proteomics, clinical proteomics exclusively considers its biomedical applications. It evaluates, often by high-throughput comparative platforms, the protein and peptide variations in body fluids, cells and tissues under different physiological and pathological conditions with the aim of discovering disease biomarkers. Among the available analytical methodologies, mass spectrometry in coupling with liquid chromatography or capillary electrophoresis demonstrated to be the eligible technique for protein detection and identification. This review summarizes the most recent applications of capillary electrophoresis-mass spectrometry to clinical proteomics, focusing on capillary zone electrophoresis separation mode and ESI and MALDI ionizations, which are the most frequently applied capillary electrophoresis-mass spectrometry hyphenated techniques., (Copyright 2010 Elsevier B.V. All rights reserved.)
- Published
- 2010
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25. Capillary electrophoresis-mass spectrometry for the analysis of amino acids.
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Desiderio C, Iavarone F, Rossetti DV, Messana I, and Castagnola M
- Subjects
- Amino Acids analysis, Electrophoresis, Capillary, Mass Spectrometry
- Abstract
In this review, the recent contribution of CE-MS technology to the analysis of amino acids, as well as the advantages of the hyphenation and the technologies involved in the instrumental coupling are reported. Different sections are dedicated to the recent contributions of CE-MS to the analysis of protein amino acids and their post-translational modifications, such as phosphorylation and sulfation. CE-MS analysis of some amino acid derivatives, such as the free methylated-derivatives of arginine is also discussed. A section is specifically devoted to the CE-MS applications in the field of chiral separation of D- and L-amino acid enantiomers.
- Published
- 2010
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26. Analysis of arginine and methylated metabolites in human plasma by field amplified sample injection capillary electrophoresis tandem mass spectrometry.
- Author
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Desiderio C, Rossetti DV, Messana I, Giardina B, and Castagnola M
- Subjects
- Acetonitriles chemistry, Arginine chemistry, Humans, Linear Models, Methylation, Reproducibility of Results, Sensitivity and Specificity, omega-N-Methylarginine chemistry, Acetonitriles blood, Arginine analogs & derivatives, Arginine blood, Electrophoresis, Capillary methods, Flow Injection Analysis methods, Tandem Mass Spectrometry methods, omega-N-Methylarginine blood
- Abstract
A CE ion trap tandem MS method was optimised for the analysis of arginine, monomethyl- and (symmetric and asymmetric) dimethylarginines in human plasma after a very reduced sample pretreatment step involving a simple protein precipitation with ACN. Several parameters affecting the analytes MS ionization and the capillary electrophoretic separation were carefully studied and optimised. The complete separation of arginine, monomethylarginine and symmetric and asymmetric dimethylarginine was obtained in formic acid BGE in short analysis time with high specificity due to MS(2) detection of specific analytes fragments. In order to achieve the detection sensitivity suitable for the analysis of asymmetric and symmetric dimethylarginine in human plasma, the field amplified sample injection was applied. Due to stacking effects, this methodology allowed to operate a consistent on-line preconcentration of the analytes before running the electrophoresis. The method was validated for linearity, repeatability, recovery and accuracy and applied to the quantitative analysis of arginine, monomethyl- and dimethylarginines in human plasma of healthy subjects.
- Published
- 2010
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27. N-acetyl cysteine directed detoxification of 2-hydroxyethyl methacrylate by adduct formation.
- Author
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Nocca G, D'Antò V, Desiderio C, Rossetti DV, Valletta R, Baquala AM, Schweikl H, Lupi A, Rengo S, and Spagnuolo G
- Subjects
- 3T3 Cells, Acetylcysteine chemistry, Animals, Cell Death drug effects, Cell Survival drug effects, Chromatography, High Pressure Liquid, Electrophoresis, Capillary, Extracellular Space drug effects, Extracellular Space metabolism, Fibroblasts cytology, Fibroblasts drug effects, Fibroblasts metabolism, Intracellular Space drug effects, Intracellular Space metabolism, Mass Spectrometry, Mice, Acetylcysteine pharmacology, Methacrylates chemistry, Methacrylates toxicity
- Abstract
Cytotoxicity of the dental resin monomer 2-hydroxyethyl methacrylate (HEMA) and the protective effects of N-acetyl cysteine (NAC) on monomer-induced cell damage are well demonstrated. The aim of our study was to analyze the hypothesis that the protection of NAC from HEMA cytotoxicity might be due to direct NAC adduct formation. To this end, using HPLC we first measured the actual intracellular HEMA concentrations able to cause toxic effects on 3T3-fibroblasts and then determined the decrease in intracellular and extracellular HEMA levels in the presence of NAC. In addition, by capillary electrophoresis coupled with mass spectrometry analysis (CE-MS), we evaluated NAC-HEMA adduct formation. HEMA reduced 3T3 cell vitality in a dose- and time-dependent manner. The concentration of HEMA inside the cells was 15-20 times lower than that added to the culture medium for cell treatment (0-8 mmol/L). In the presence of 10 mmol/L NAC, both intracellular and extracellular HEMA concentrations greatly decreased in conjunction with cytotoxicity. NAC-HEMA adducts were detected both in the presence and absence of cells. Our findings suggest that the in vitro detoxification ability of NAC against HEMA-induced cell damage occurs through NAC adduct formation. Moreover, we provide evidence that the actual intracellular concentration of HEMA able to cause cytotoxic effects is at least one magnitude lower than that applied extracellularly., (Copyright 2009 Elsevier Ltd. All rights reserved.)
- Published
- 2010
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28. Enantiomeric separation of baclofen by capillary electrophoresis tandem mass spectrometry with sulfobutylether-beta-cyclodextrin as chiral selector in partial filling mode.
- Author
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Desiderio C, Rossetti DV, Perri F, Giardina B, Messana IC, and Castagnola M
- Subjects
- Electrophoresis, Capillary instrumentation, Formates chemistry, Pharmaceutical Preparations chemistry, Sensitivity and Specificity, Stereoisomerism, beta-Cyclodextrins, Baclofen isolation & purification, Electrophoresis, Capillary methods, Tandem Mass Spectrometry methods
- Abstract
Capillary electrophoresis (CE) coupled to tandem mass spectrometry was applied to the chiral separation of baclofen using sulfobutylether-beta-cyclodextrin chiral selector in partial filling counter current mode. On-line UV detection was simultaneously used. Method optimization was performed by studying the effect of cyclodextrin and BGE concentration as well as sheath liquid composition on analyte migration time and enantiomeric resolution. The cyclodextrin showed stereoselective complexation towards baclofen enantiomers, allowing chiral resolution at low concentration. The CE capillary protrusion from the ESI needle relevantly affected the chiral resolution and the analyte migration time. Complete enantiomeric separation was obtained by using 0.25 M formic acid BGE containing 1.75 mM of chiral selector and water/methanol (30:70, v/v) 3% formic acid as sheath liquid. The method exhibited a LOD of 0.1 microg/mL (racemic concentration) in MS3 product ion scan mode of detection and was applied to the analysis of racemic baclofen in pharmaceutical formulations.
- Published
- 2008
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29. Optimization of a rapid capillary electrophoresis ESI-IT tandem mass spectrometry method for the analysis of short-chain carnitines in human plasma.
- Author
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Desiderio C, De Rossi A, Inzitari R, Mancinelli A, Rossetti DV, Castagnola M, and Messana I
- Subjects
- Humans, Molecular Structure, Time Factors, Carnitine blood, Carnitine chemistry, Electrophoresis, Capillary methods, Tandem Mass Spectrometry methods
- Abstract
A capillary electrophoresis (CE) method coupled to electrospray ionization ion trap tandem mass spectrometry (ESI-IT-MS/MS) is described for the rapid analysis of carnitine, acetylcarnitine, and propionylcarnitine in human plasma. Optimization of the procedure was achieved by a reduced sample pretreatment and after examining several physicochemical parameters that influence both the CE separation and the MS analytes detection. The analysis of total carnitine in human plasma after hydrolysis of short-chain metabolites is also shown. The analysis of carnitine and metabolites was obtained in less than 10 min using a 200 mM ammonium formate buffer, pH 2.5, with high sensitivity and specificity using the MS detection in product ion scan mode. The method was tested for quantitative recovery using dialyzed human plasma as matrix and showed linearity in the concentrations ranges 20-160, 1-32, and 0.25-8 microM for carnitine, acetylcarnitine, and propionylcarnitine with (squared) correlation coefficients of 0.9984, 0.9995, and 0.9991, respectively. The intraday and intermediate analysis repeatability and accuracy are within 15% of relative standard deviation (RSD) at low, medium, and high concentration and within/or slight exceeding 20% at the lower limit of quantitation (LLOQ). The method is sensitive for determining carnitine and its metabolites in human plasma with high specificity.
- Published
- 2008
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30. Rapid determination of short chain carnitines in human plasma by electrospray ionisation-ion trap mass spectrometry using capillary electrophoresis instrument as sampler.
- Author
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Desiderio C, Mancinelli A, De Rossi A, Rossetti DV, Inzitari R, Messana I, Giardina B, and Castagnola M
- Subjects
- Carnitine chemistry, Humans, Molecular Structure, Reproducibility of Results, Carnitine blood, Electrophoresis, Capillary methods, Spectrometry, Mass, Electrospray Ionization methods
- Abstract
A capillary electrophoresis apparatus was used as sampler for flow injection analysis (FIA) in tandem mass spectrometry of L-carnitine and its acetyl- and propionyl-metabolites in human plasma. The capillary electrophoresis instrument was coupled to the ion trap mass spectrometer by an electrospray ionization coaxial sheath liquid interface. The electrophoresis capillary introduced the sample directly into the source by applying a prolonged sample injection. The use of the capillary electrophoresis apparatus miniaturised the FIA procedure, substantially reducing the quantities of solvents and samples used, and allowed rapid automated sequential analyses. The method was optimised and validated using a dialyzed human plasma matrix. The plasma samples were analysed after a simple, rapid deproteinisation procedure with acetonitrile and diluted 70 times before direct injection into the mass spectrometer for product ion scan MS/MS analysis in positive ionisation. The total analysis time was 5 min, including capillary preconditioning and acquisition time (3 min). The method was sensitive, allowing the determination of L-, L-acetyl- and L-propionyl-carnitines at 140, 14 and 3.6 nM concentrations (injected values) corresponding to lower limit of quantitation values in plasma of 10, 1 and 0.25 microM, respectively. The method was processed for full validation and applied to the analysis of L-carnitine and its short chain derivatives in human plasma samples.
- Published
- 2007
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31. Detection in human saliva of different statherin and P-B fragments and derivatives.
- Author
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Inzitari R, Cabras T, Rossetti DV, Fanali C, Vitali A, Pellegrini M, Paludetti G, Manni A, Giardina B, Messana I, and Castagnola M
- Subjects
- Adult, Amino Acid Sequence, Chromatography, High Pressure Liquid, Humans, Molecular Sequence Data, Spectrometry, Mass, Electrospray Ionization, Tandem Mass Spectrometry, Peptide Fragments analysis, Saliva chemistry, Salivary Proteins and Peptides analysis
- Abstract
Statherin is a multifunctional polypeptide specific of human saliva involved in oral calcium homeostasis, phosphate buffering and formation of protein networks. Salivary P-B peptide is usually included into the basic proline-rich protein family but it shows some similarities with statherin and its specific biological role is still undefined. In this study, various fragments and derivatives of statherin and P-B peptide were consistently detected by RP-HPLC ESI-IT MS in 23 samples of human saliva. They were: statherin mono- and non-phosphorylated, statherin Des-Phe(43) (statherin SV1), statherin Des-Thr(42),Phe(43), statherin Des-Asp(1), statherin Des(6-15) (statherin SV2), statherin Des(1-9), statherin Des(1-10), statherin Des(1-13) and P-B Des(1-5). Statherin SV3 (statherin Des(6-15), Phe(43)) was detected only in one sample. Identity of the fragments was confirmed either by MS/MS experiments or by enzymatic digestion or by Edman sequencing. Detection of the fragments suggests that statherin and P-B peptide are submitted to post-translational proteolytic cleavages that are common to other classes of salivary proteins.
- Published
- 2006
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32. A cascade of 24 histatins (histatin 3 fragments) in human saliva. Suggestions for a pre-secretory sequential cleavage pathway.
- Author
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Castagnola M, Inzitari R, Rossetti DV, Olmi C, Cabras T, Piras V, Nicolussi P, Sanna MT, Pellegrini M, Giardina B, and Messana I
- Subjects
- Amino Acid Sequence, Binding Sites, Endopeptidases metabolism, Histatins, Humans, Mass Spectrometry, Proteins analysis, Saliva chemistry, Peptide Fragments analysis, Salivary Proteins and Peptides analysis
- Abstract
The systematic search by tandem mass spectrometry of human saliva from four different subjects, of 136 possible fragments originated from histatin 3, allowed the detection of 24 different peptides. They include, with the exception of histatin 4, all the known histatin 3 fragments, namely histatins 5-12 and the peptides corresponding to 15-24, 26-32, 29-32 residues, and 13 new fragments corresponding to 1-11, 1-12, 1-13, 5-13, 6-11, 6-13, 7-11, 7-12, 7-13, 14-24, 14-25, 15-25, and 28-32 residues of histatin 3. On the contrary, none of 119 possible fragments of histatin 1, including histatin 2, was detected. The results suggest that the genesis of histatin 3-related peptides, being under the principal action of trypsin-like activities, is probably not a random process but rather follows a sequential fragmentation pathway. Lack of detection of C-terminal fragments, with the exception of 26-32, 28-32, and 29-32 fragments, suggested that arginine 25 should be the first cleavage site, generating histatin 6 and 26-32 fragments. The genesis of 28-32 and 29-32 fragments and histatin 5 should implicate a subsequent exo-protease action. Similarly, lack of detection of fragments having Lys-5 and Arg-6 at the N terminus and Arg-25 at the C terminus strongly suggested that sequences KRKF (11-14 residues) and AKR (4-6 residues) should be the second and the third cleavage sites, respectively. Lys-17 and Arg-22 are not cleaved at all.
- Published
- 2004
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33. Different binding thermodynamics of Ni2+, Cu2+, and Zn2+ to bacitracin A1 determined by capillary electrophoresis.
- Author
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Castagnola M, Rossetti DV, Inzitari R, Lupi A, Zuppi C, Cabras T, Fadda MB, Onnis G, Petruzzelli R, Giardina B, and Messana I
- Subjects
- Bacitracin metabolism, Cations, Divalent chemistry, Cations, Divalent metabolism, Copper metabolism, Hydrogen-Ion Concentration, Molecular Structure, Nickel metabolism, Protons, Thermodynamics, Zinc metabolism, Bacitracin chemistry, Copper chemistry, Electrophoresis, Capillary methods, Nickel chemistry, Zinc chemistry
- Abstract
Thermodynamics of the binding of Ni(2+), Cu(2+) and Zn(2+) to bacitracin A(1) was studied by capillary electrophoresis measuring the peptide effective mobility at different pH in the presence of increasing concentration of the three ligands. The affinity follows the order Ni(2+) > Cu(2+) > Zn(2+), with association constant values of (2.3 +/- 0.1)x10(4), (4.9 +/- 0.2)x10(3), and (1.5 +/- 0.1)x10(3) M(-1), respectively. The only model able to rationalize mobility data implies that metal ion binds to the P(0) peptide form. Moreover, mobility values indicated a change of bacitracin A(1) acidic properties on Ni(2+) and Cu(2+) binding, with a shift of the pK(a) of N-terminal Ile-1 from 7.6 to about 5 and of the pK(a) of the delta-amino group of D-Orn-7 from 9.7 to about 7. Even though on Zn(2+) binding a shift of the N-terminal Ile-1 pK(a) was observed, restrictions in the pH range suitable for investigation, due to precipitation phenomena, did not allow establish if the shift of D-Orn-7 lateral chain pK(a) also occurred. Nonetheless, if present, the shift should be limited to the 7.8-9.7 range. Mobility data indicated that the Stokes radius of the complexes is ca. 3 A lower than that of the free peptide. The present results indicate that metal-ion binding to bacitracin A(1) is more complex than previously assumed.
- Published
- 2004
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34. Determination of urinary hippuric acid by micellar electrokinetic capillary chromatography.
- Author
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Zuppi C, Rossetti DV, Vitali A, Vincenzoni F, Giardina B, Castagnola M, and Messana I
- Subjects
- Creatinine urine, Glomerular Filtration Rate, Magnetic Resonance Spectroscopy, Reproducibility of Results, Chromatography, Micellar Electrokinetic Capillary methods, Hippurates urine
- Abstract
We propose a method for the simultaneous determination of hippuric acid (HA) and creatinine based on capillary micellar electrokinetic chromatography. Experimental conditions were 20 mM sodium phosphate, pH 7.20, 25 mM sodium dodecyl sulfate, 5% (v/v) acetonitrile. Electropherograms evidenced HA and creatinine peaks in less than 12 min. The method showed good linearity for both analytes and satisfactory within-day precision. The present method, which is accurate, sensitive, rapid and simple, may be applied to single-spot urine samples.
- Published
- 2003
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35. Capillary electrophoretic study of the binding of zinc(II) ion to bacitracin A1 in water-2,2,2-trifluoroethanol.
- Author
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Castagnola M, Rossetti DV, Inzitari R, Vitali A, Lupi A, Zuppi C, Cabras T, Fadda MB, Podda I, Petruzzelli R, Giardina B, and Messana I
- Subjects
- Water chemistry, Bacitracin chemistry, Electrophoresis, Capillary methods, Trifluoroethanol chemistry, Zinc chemistry
- Abstract
Binding of Zn(2+) to bacitracin A(1) was studied by capillary electrophoresis in water/2,2,2-trifluoroethanol (70/30 v/v) at different apparent pH values in order to estimate the association constant of metal, the acidic dissociation constants and the Stokes radii of both free and bounded peptide in apolar environment. The Stokes radii of the free peptide species were compared with those in aqueous solution, as obtained in a recent study performed by our group, indicating that apolar environment stabilizes bacitracin A(1) in a conformational structure with the lateral chain of apolar amino acids exposed on the external surface. This conformation of the macrocyclic dodecapeptide is ready to interact with Zn(2+) ion, as pointed out by the strong increase of the association constant measured in water/2,2,2-trifluoroethanol with respect to the value obtained in aqueous solution. In addition, whereas Zn(2+) ion binding in aqueous solution provides a sensible reduction of peptide Stokes radius, no sensible variations following to ion binding were observed in hydro-organic solution. The present results suggest that the apolar environment, rather than the metal ion binding, could be responsible for the conformational transition that brings bacitracin A(1) towards its biologically active structure.*
- Published
- 2003
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36. Determination of the post-translational modifications of salivary acidic proline-rich proteins.
- Author
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Castagnola M, Cabras T, Inzitari R, Zuppi C, Rossetti DV, Petruzzelli R, Vitali A, Loy F, Conti G, and Fadda MB
- Subjects
- Amino Acid Sequence, Chromatography, High Pressure Liquid, Humans, Mass Spectrometry, Molecular Weight, Peptides isolation & purification, Peptides metabolism, Phosphorylation, Proline chemistry, Protein Isoforms chemistry, Pyrrolidonecarboxylic Acid chemistry, Salivary Proteins and Peptides drug effects, Serine chemistry, Serine metabolism, Spectrometry, Mass, Electrospray Ionization, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Trypsin metabolism, Peptides chemistry, Protein Processing, Post-Translational, Salivary Proteins and Peptides chemistry
- Abstract
Human salivary acidic proline-rich proteins were analyzed by electrospray-ion trap mass spectrometry and by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. All acidic-PRP isoforms share a common N-terminal region, which contains a pyroglutamic acid residue at the N-terminus, and two phosphorylation sites on Ser 8 and 22. At the same time, HPLC-MS spectra revealed isoforms of PRP-1 and PRP-3 having a different number of phosphoserine residues, namely, a mono-phosphorylated form of PRP-1 and PRP-3 and a tri-phosphorylated form of PRP-1. The analysis of the masses of tryptic digests suggested that the third phosphate residue should be located on Ser 17. Another protein with a mass of 30,923 amu was detected along the HPLC pattern and MS data of its tryptic digest suggested that it corresponds to the dimer of Pa, the isoform of PRP-1 with a substitution Arg-Cys at 103 position. Finally, structural identification is pending for another post-translational modification of acidic-PRP that provides an increase of 111-114 amu.
- Published
- 2003
- Full Text
- View/download PDF
37. Affinity capillary electrophoresis study of the linkage existing between proton and zinc ion binding to bacitracin A1.
- Author
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Castagnola M, Rossetti DV, Inzitari R, Vitali A, Lupi A, Zuppi C, Cabras T, Fadda MB, Petruzzelli R, Giardina B, and Messana I
- Subjects
- Bacitracin analysis, Buffers, Cations, Divalent, Hydrogen-Ion Concentration, Mathematics, Molecular Structure, Osmolar Concentration, Protein Binding, Time Factors, Bacitracin chemistry, Electrophoresis, Capillary methods, Protons, Zinc chemistry
- Abstract
Measurements by capillary electrophoresis (CE) of bacitracin A(1) effective mobility at different pH values permitted to estimate the five acidic dissociation constants and the Stokes radii at different protonation stages of the macrocyclic dodecapeptide. The pK(a) values were 3.6 and 4.4 for the two carboxylic groups of the lateral chains of D-Asp-11 and D-Glu-4, respectively, 6.4 for the aza-atom of the imidazole ring of His-10, 7.6 for the amino group of N-terminal Ile-1 and 9.7 for the delta-amino group of D-Orn-7, very close to the values obtained by other researchers by titration experiments. In agreement with a rigid macrocyclic structure the Stokes radii of different protonated forms ranged only between 14.3 and 14.8 A. Best fitting procedures performed on experimental mobility measured at two different pH values (5.50 and 6.72) in the presence of increasing Zn(+2) concentration allowed confirming the model that assumes the binding of Zn(+2) to P(0) peptide form with a 1.5 x 10(3) M(-1) intrinsic association constant. Following to Zn(+2) binding, the pK(a) of the amino group of N-terminal Ile-1 is shifted from 7.6 to 5.9 and the Stokes radius is reduced of about 3 A. The mean charge of the bacitracin A(1)-Zn(+2) complex resulted +1.67 and +1.12 at pH 5.50 and 6.72, respectively. These results suggest that the amino group of N-terminal Ile-1 is not essential for Zn(+2) binding.
- Published
- 2003
- Full Text
- View/download PDF
38. Characterization of dendrimer properties by capillary electrophoresis and their use as pseudostationary phases.
- Author
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Castagnola M, Zuppi C, Rossetti DV, Vincenzoni F, Lupi A, Vitali A, Meucci E, and Messana I
- Subjects
- Dendrimers, Chromatography, Micellar Electrokinetic Capillary methods, Electrophoresis, Capillary methods, Polyamines
- Abstract
The general properties of dendrimers and in particular their electrolytic characteristics that are relevant in electrokinetic separations, are described. In order to confirm theoretical considerations on commercial dendrimer charge and hydrodynamic radius, several capillary zone electrophoresis (CZE) experiments were performed. Electrophoretic mobilities measured at different pH values indicated a sensible increase of dendrimer hydrodynamic radius at pH values lower than 2.5. This was probably due to the Coulombic repulsion of charged amine groups of the inner dendrimer shells. The principal reasons that should address the use of dendrimers as pseudostationary phases in micellar electrokinetic chromatography (MEKC) are discussed. Moreover, a survey of different separations performed utilizing dendrimers in MEKC as well as of several future plausible uses of various classes of dendrimers is presented.
- Published
- 2002
- Full Text
- View/download PDF
39. Determination of S-nitrosoglutathione in erythrocytes by capillary zone electrophoresis.
- Author
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Messana I, Rossetti DV, Misiti F, Vincenzoni F, Tellone E, Giardina B, and Castagnola M
- Subjects
- Electrophoresis, Capillary methods, Glutathione blood, Glutathione chemistry, Glutathione Disulfide chemistry, Humans, S-Nitrosoglutathione, Erythrocytes chemistry, Glutathione analogs & derivatives, Nitroso Compounds blood
- Abstract
A method for separation and quantification of S-nitrosoglutathione in red cell extracts by capillary electrophoresis is reported. The method is based on the direct analysis of the metaphosphoric acid erythrocyte extract containing diethylenetriaminepentaacetic acid. Optimization of the method is briefly discussed. Best results in the shortest time were obtained at 25 degrees C, using a coated capillary, 7 kV applied voltage and phosphate sodium 40 mmol/L (pH 2.2) as running buffer. Reproducibility, detection limits, and recoveries of S-nitrosoglutathione analyses were checked. The results evidenced that S-nitrosoglutathione is formed in erythrocytes treated with S-nitrosocysteine, a transnitrosating agent. Under our experimental conditions, the contemporaneous detection and quantification of reduced and oxidized glutathione present in cell extract could also be performed.
- Published
- 2000
- Full Text
- View/download PDF
40. Determination of biophysical parameters of polypeptide retro-inverso isomers and their analogues by capillary electrophoresis.
- Author
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Hearn MT, Keah HH, Boysen RI, Messana I, Misiti F, Rossetti DV, Giardina B, and Castagnola M
- Subjects
- Amino Acid Sequence, Biophysical Phenomena, Biophysics, Electrophoresis, Capillary, Indicators and Reagents, Isomerism, Molecular Sequence Data, Peptides chemistry
- Abstract
The relationship between the electrophoretic mobility, microobs, Stokes radius, rs, ionization state, and solution conformation of the all L-alpha-polypeptide, 1, the corresponding retro-all D-alpha-polypeptide, 2, and several truncated analogues, 3-5, has been investigated under low pH buffer conditions by high-performance capillary zonal electrophoresis (HPCZE) with coated capillaries. The results confirm that, under these conditions, the all L-alpha-polypeptide, 1, and its retro-inverso isomer, 2, exhibit nonidentical electrophoretic mobilities and thus different Stokes radii. At higher pH values, i.e., pH 5.0, the electrophoretic behavior of this retro-inverso isomer pair, however, converges. These results indicate that variations in the dipole characteristics of the polypeptide main chain and subtle differences introduced by the spatial constraints of the L-alpha-Pro-->D-alpha-Pro residue replacement lead to differences in the Stokes radii and electrophoretic mobilities of these polypeptides. Since the observed electrophoretic mobilities, microobs, reflect the mean of the mobilities of each charge species participating according to their Stokes radius or their intrinsic charge and mole fraction abundances, the results confirm that polypeptide retro-inverso isomers with unmodified amino and carboxy termini are resolvable. This outcome was achieved despite their notional topographical and conformational similarities as assessed from high-field proton nuclear magnetic resonance (1H NMR) spectroscopy and circular dichroism (CD) spectroscopy.
- Published
- 2000
- Full Text
- View/download PDF
41. The pH dependence of predictive models relating electrophoretic mobility to peptide chemico-physical properties in capillary zone electrophoresis.
- Author
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Castagnola M, Rossetti DV, Corda M, Pellegrini M, Misiti F, Olianas A, Giardina B, and Messana I
- Subjects
- Amino Acid Sequence, Chemical Phenomena, Chemistry, Physical, Hydrogen-Ion Concentration, Molecular Sequence Data, Osmosis, Electrophoresis, Capillary, Models, Chemical, Peptides chemistry
- Abstract
We applied best fitting procedures to capillary electrophoresis (CE) mobility values, measured at varying acidic pH, of a set of 21 peptides with a molecular mass ranging from about 350 to 1850 Da. This method allowed the contemporary measurements of C-terminus and carboxylic group of the side-chain of aspartic and glutamic acid dissociation constants and of peptide Stokes radius at different protonation stages. Stokes radius was related to peptide molecular mass M at the power of a fractional coefficient, and best correlation was found at pH 2.25, the fractional coefficient being equal to 0.68. This value is close to that proposed by R. E. Offord (Nature 1966, 211, 591-593), who suggested a proportionality between the polymer Stokes radius and M(2/3). The coefficient value decreases at higher pH, reaching a value of 0.58 at pH 4.25, corresponding to a mean peptide conformational transition towards more compact structures as a consequence of C-terminus dissociation. The measurement of the dissociation constants of each peptide allowed us to determine the percentage error on peptide charge predictions performed utilizing mean dissociation constants. Even for the charge, the best predictive performance is obtained at the most acidic edge of the range of the pH studied, mainly at pH 2.25. Conclusively, this study shows that the best performance of predictive models for peptide CE mobility is obtainable in the very acidic pH range (2.25-2.50) and in the absence of electroosmotic flow, and that a satisfactory predictive equation of peptide electrophoretic mobility (m2V(-1)s(-1) is given by mu = 85.4(Z/M(0.68))10(-8).
- Published
- 1998
- Full Text
- View/download PDF
42. Predictive model for capillary electrophoretic peptide mobility in 2,2,2-trifluoroethanol-water solution.
- Author
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Castagnola M, Rossetti DV, Corda M, Pellegrini M, Misiti F, Olianas A, Giardina B, and Messana I
- Subjects
- Mathematical Computing, Solutions, Electrophoresis, Capillary methods, Models, Molecular, Peptides analysis, Trifluoroethanol, Water
- Abstract
Using capillary electrophoresis (CE) on a set of 21 peptides with a molecular mass ranging from about 350 to 1850 Da, the Stokes radii at different protonation stages and the acidic dissociation constants in water and in a 2,2,2-trifluoroethanol (TFE) water mixture (30% v/v) were determined. These results permitted us to establish separately the reliability of semiempirical models utilized for the prediction of peptide size and charge at different acidic pHapp (pHapp range: 2.00-4.25). The data obtained on size and charge were utilized in order to provide suitable mobility predictions on the basis of the charge-to-size ratio. The best predictive conditions for size and charge were found at the most acidic range of pHapp studied (2.00-2.25), either in water or a TFE-water mixture, and reliable predictive equations for peptide mobility were established at this pHapp.
- Published
- 1998
- Full Text
- View/download PDF
43. Peptide analysis by capillary (zone) electrophoresis.
- Author
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Messana I, Rossetti DV, Cassiano L, Misiti F, Giardina B, and Castagnola M
- Subjects
- Buffers, Hydrogen-Ion Concentration, Models, Chemical, Peptides chemistry, Sensitivity and Specificity, Electrophoresis, Capillary methods, Peptides analysis
- Abstract
In this review various aspects concerning the application of capillary (zone) electrophoresis for peptide analysis will be critically examined. First, the basic instrumental requirements of CE apparatus and the strategies employed to enhance sensitivity in the analysis of underivatized sample are described. Multidimensional separative techniques of complex peptide mixtures that use CE as final step and the coupling of CE with mass spectrometry are subsequently discussed. A theoretical section describes the relationships existing between peptide mobility and the pH of the separation buffer. These relationships evidence that proton dissociation constants and Stokes radius at different protonation stages can be calculated by measuring the electrophoretic mobility at different pH values. Investigation of peptide mobility dependence on pH allows us to establish the optimum conditions, in terms of resolution, for peptide separation. Subsequently, a critical discussion about semiempirical models predicting peptide mobility as a function of chemico-physical peptide properties is presented. A section is devoted to the description of principles of peptide affinity capillary electrophoresis, underlining the similarity with peptide-proton interaction. CE separations performed in aquo-organic solvents are also critically discussed, showing the good performance obtained by using water-2,2,2-trifluoroethanol solutions. Finally, selected CE applications for the determination of peptide chemico-physical properties and conventional analysis, like peptide mapping, are reported.
- Published
- 1997
- Full Text
- View/download PDF
44. Determination of peptide dissociation constants and Stokes radius at different protonation stages by capillary electrophoresis.
- Author
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Castagnola M, Rossetti DV, Cassiano L, Misiti F, Pennacchietti L, Giardina B, and Messana I
- Subjects
- Adrenocorticotropic Hormone chemistry, Amino Acid Sequence, Bradykinin chemistry, Chemical Phenomena, Chemistry, Physical, Enkephalin, Leucine chemistry, Enkephalin, Methionine chemistry, Hydrogen-Ion Concentration, Mathematics, Peptide Fragments chemistry, Electrophoresis, Capillary methods, Peptides chemistry, Protons
- Abstract
Peptide electrophoretic mobility measured by capillary zone electrophoresis can be regarded as deriving from the mean of mobilities of different protonated forms, each one participating according to its charge. Stokes radius and relative percentage. The percentage is a function of the peptide dissociation constant and solution pH. Therefore, mobility modifications due to pH variations can be related to peptide dissociation constant, charge, and Stokes radius throughout general binding equations. Thus, not only can peptide dissociation constants be measured, but information about Stokes radius modifications linked to proton loss can also be obtained with picomoles of peptide.
- Published
- 1996
- Full Text
- View/download PDF
45. Effect of 2,2,2-trifluoroethanol on capillary zone electrophoretic peptide separations.
- Author
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Castagnola M, Cassiano L, Messana I, Paci M, Rossetti DV, and Giardina B
- Subjects
- Adrenocorticotropic Hormone chemistry, Adrenocorticotropic Hormone isolation & purification, Amino Acid Sequence, Chemical Phenomena, Chemistry, Physical, Electrophoresis, Capillary statistics & numerical data, Enkephalin, Leucine chemistry, Enkephalin, Leucine isolation & purification, Enkephalin, Methionine chemistry, Enkephalin, Methionine isolation & purification, Hydrogen-Ion Concentration, Molecular Sequence Data, Myoglobin chemistry, Myoglobin isolation & purification, Peptide Fragments chemistry, Peptide Fragments isolation & purification, Peptides chemistry, Electrophoresis, Capillary methods, Peptides isolation & purification, Trifluoroethanol pharmacology
- Abstract
The use of 2,2,2-trifluoroethanol-water mixtures for peptide separations by capillary zone electrophoresis (CZE) displays some advantages over aqueous solutions. First, the increase in viscosity reduces and stabilizes the running current and facilitates heat dispersion, with a consequent improvement in the number of theoretical plates. Second, the decrease in the dielectric constant leads to a modification of the dissociation constants of the ionizable groups. The consequence is a change in selectivity that, for several favourable peptide pairs, provides an increase in resolution. Third, the interaction trifluoroethanol with the peptide modifies the Stokes radius in a manner strongly dependent on the peptide sequence. This can also be utilized for an increase in CZE performance. Fourth, the structural properties of 2,2,2-trifluoroethanol are particularly useful for an improvement in the separation of large apolar peptides. Finally, the use of trifluoroethanol strongly stabilizes the capillary coating.
- Published
- 1996
- Full Text
- View/download PDF
46. The use of capillary electrophoresis for the determination of hemoglobin variants.
- Author
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Castagnola M, Messana I, Cassiano L, Rabino R, Rossetti DV, and Giardina B
- Subjects
- Amino Acids analysis, Chromatography methods, Chromatography, High Pressure Liquid, Electrochemistry, Globins isolation & purification, Hemoglobin A analysis, Hemoglobin A metabolism, Hemoglobin C analysis, Hemoglobin C metabolism, Hemoglobin, Sickle analysis, Hemoglobin, Sickle metabolism, Hemoglobins, Abnormal metabolism, Humans, Macromolecular Substances, Micelles, Peptide Fragments analysis, Peptide Fragments metabolism, Peptide Mapping, Trypsin metabolism, Electrophoresis, Capillary methods, Hemoglobins, Abnormal analysis
- Abstract
The application of capillary electrophoresis and related techniques for the detection of hemoglobin variants is described. Capillary zone electrophoresis (CZE) was applied for the analysis of intact tetrameric hemoglobin. CZE under denaturing conditions was used for the separation of globin chains. Both CZE and micellar electrokinetic capillary chromatography were applied for a fast and sensitive separation of tryptic digests and for the analysis of amino acid derivatives.
- Published
- 1995
- Full Text
- View/download PDF
47. Capillary zone electrophoresis of peptides: prediction of the electrophoretic mobility and resolution.
- Author
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Castagnola M, Cassiano L, Messana I, Nocca G, Rabino R, Rossetti DV, and Giardina B
- Subjects
- Amino Acid Sequence, Electrophoresis, Polyacrylamide Gel, Enkephalin, Leucine isolation & purification, Enkephalin, Methionine isolation & purification, Hydrogen-Ion Concentration, Indicators and Reagents, Molecular Sequence Data, Peptides chemistry, Peptides isolation & purification
- Abstract
The determination of the pKa values of some selected peptides of similar size was performed by microtitration, which makes possible an accurate determination of the peptide charge as a function of the solution pH. Capillary zone electrophoresis separation of these peptides on modified capillaries at acidic pH showed that the electrophoretic mobility correlates with the peptide charge. This observation suggests that when an appropriate charge value is used, the basic electrophoretic equation is respected and, at least at a peptide charge value less than 1, the utilization of alternative semi-empirical predictions is not necessary. As a general rule, a peptide separation at acidic pH values is to be preferred to that at basic pH values. In fact, at basic pH a separation in the absence of both electroosmotic flow and of spurious interactions between the peptides and the inner wall of the capillary is difficult, owing to the instability of capillary modification. Further, from the differences in the peptide charge, a prediction of the best resolution as a function of the pH could be obtained; in fact, the resolution, for peptides of similar size and in the absence of electroosmotic flow, is connected to a simple equation, where the principal term depends on the effective charge of the peptides, which is a function of the pH of the solution and the pKa values of the peptides. The predictions of resolution at acidic pH agreed well with the experimental results; the spatial resolution measured in the separation of met- and leu-enkephalin was virtually coincident with the predicted resolution; in the case of a mixture of four model tetrapeptides of sequence GGNA, GGQA, GGDA and GGEA some anomalous results with respect to the predicted resolutions were observed. Nevertheless, an acceptable prediction can also be made in this case.
- Published
- 1994
- Full Text
- View/download PDF
48. Separation of reduced and oxidized glutathione by micellar electrokinetic capillary chromatography.
- Author
-
Castagnola M, Di Pierro D, Scatena R, Tavazzi B, Nocca G, Rossetti DV, and Giardina B
- Subjects
- Electrophoresis, Micelles, Oxidation-Reduction, Glutathione isolation & purification
- Abstract
The separation of reduced and oxidized glutathione at an absolute sensitivity of about 100 pg by micellar electrokinetic capillary chromatography without derivatization is described. The time required for the separation is less than 10 min (the time between two following injections is about 15 min). The separation is characterized by high efficiency and good reliability. A partition mechanism is responsible for the high resolution observed. The method was utilized for the analysis of commercial preparations of glutathione and a good agreement with the expected results was obtained; the oxidation of the commercial glutathione in solution was easily analysed.
- Published
- 1993
- Full Text
- View/download PDF
49. Peptide mapping through the coupling of capillary electrophoresis and high-performance liquid chromatography: map prediction of the tryptic digest of myoglobin.
- Author
-
Castagnola M, Cassiano L, Rabino R, Rossetti DV, and Bassi FA
- Subjects
- Amino Acids analysis, Animals, Horses, Peptide Mapping, Chromatography, High Pressure Liquid methods, Electrophoresis methods, Myoglobin metabolism, Trypsin metabolism
- Abstract
The tryptic map of horse myoglobin was analysed through capillary electrophoresis using capillaries modified by a monolayer of acrylamide. The results were reproducible and the map was obtained in less than 30 min from ca. 8 pmol of tryptic digest. The peptide identification was performed using peptides previously identified by high-performance liquid chromatography. The peak areas measured using the two techniques are closely related, and the comparison of elution and migration times shows that the two techniques provide different maps. Furthermore, using the semiempirical relationship suggested by Grossman et al. [Anal. Biochem., 179 (1989) 28], which links the electrophoretic mobility to the charge of the peptide and its number of amino acids, a good agreement between predicted and experimental mobilities was observed.
- Published
- 1991
- Full Text
- View/download PDF
50. Hb K-Ibadan [beta 46(CD5)Gly----Glu] in an Italian family.
- Author
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Castagnola M, Cassiano L, Rossetti DV, Marucci L, Ferro A, Scarano C, Monaco M, and Celozzi AM
- Subjects
- Adolescent, Adult, Amino Acids analysis, Child, Child, Preschool, Glutamates genetics, Glutamic Acid, Glycine genetics, Hemoglobins, Abnormal genetics, Heterozygote, Humans, Italy, Mutation, Hemoglobins, Abnormal chemistry
- Published
- 1990
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