Publications by authors named "Vincenzo Cunsolo"

52 Publications

Quantitative Label-Free Comparison of the Metabolic Protein Fraction in Old and Modern Italian Wheat Genotypes by a Shotgun Approach.

Molecules 2021 Apr 29;26(9). Epub 2021 Apr 29.

Laboratory of Organic Mass Spectrometry, Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy.

Wheat represents one of the most important cereals for mankind. However, since wheat proteins are also the causative agent of several adverse reactions, during the last decades, consumers have shown an increasing interest in the old wheat genotypes, which are generally perceived as more "natural" and healthier than the modern ones. Comparison of nutritional value for modern and old wheat genotypes is still controversial, and to evaluate the real impact of these foods on human health comparative experiments involving old and modern genotypes are desirable. The nutritional quality of grain is correlated with its proteomic composition that depends on the interplay between the genetic characteristics of the plant and external factors related to the environment. We report here the label-free shotgun quantitative comparison of the metabolic protein fractions of two old Sicilian landraces (Russello and Timilia) and the modern variety Simeto, from the 2010-2011 and 2011-2012 growing seasons. The overall results show that Timilia presents the major differences with respect to the other two genotypes investigated. These differences may be related to different defense mechanisms and some other peculiar properties of these genotypes. On the other hand, our results confirm previous results leading to the conclusion that with respect to a nutritional value evaluation, there is a substantial equivalence between old and modern wheat genotypes. Data are available via ProteomeXchange with identifier .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/molecules26092596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8124627PMC
April 2021

Paleoproteomic profiling of organic residues on prehistoric pottery from Malta.

Amino Acids 2021 Feb 13;53(2):295-312. Epub 2021 Feb 13.

Laboratory of Organic Mass Spectrometry, Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125, Catania, Italy.

Mass spectrometry-based approaches have been successfully applied for identifying ancient proteins in bones and other tissues. On the contrary, there are relatively few examples of the successful recovery and identification of archeological protein residues from ceramic artifacts; this is because ceramics contain much lower levels of proteins which are extensively degraded by diagenetic effects. In this paper, we report the results of the characterization of proteins extracted from pottery of the Maltese site of Baħrija, the guide-site for the Baħrija period (half of 9th-second half of eighth century BCE), recently identified as the final part of the Borġ in-Nadur culture. Proteomic data here reported confirm that one of the major issue of these kind of studies is represented by contamination of animal and human agents that may complicate endogenous protein identification and authentication. The samples tested included a small group of ceramic forms, namely three tableware and six coarse ware thought to have been used in food preparation and/or storage. In this context, the limited availability of paleobotanical and archeozoological analyses may be compensated by the outcomes of the first proteomics profiling which, even if obtained on a limited selection of vessels, revealed the centrality of wheat in the diet of the ancient community of Baħrija. The data have been deposited to the ProteomeXchange with identifier < PXD022848 > .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00726-021-02946-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7910365PMC
February 2021

Post-Translational Modification Analysis of VDAC1 in ALS-SOD1 Model Cells Reveals Specific Asparagine and Glutamine Deamidation.

Antioxidants (Basel) 2020 Dec 2;9(12). Epub 2020 Dec 2.

Department of Biological, Geological and Environmental Sciences, Molecular Biology Laboratory, University of Catania, Via S. Sofia 64, 95123 Catania, Italy.

Mitochondria from affected tissues of amyotrophic lateral sclerosis (ALS) patients show morphological and biochemical abnormalities. Mitochondrial dysfunction causes oxidative damage and the accumulation of ROS, and represents one of the major triggers of selective death of motor neurons in ALS. We aimed to assess whether oxidative stress in ALS induces post-translational modifications (PTMs) in VDAC1, the main protein of the outer mitochondrial membrane and known to interact with SOD1 mutants related to ALS. In this work, specific PTMs of the VDAC1 protein purified by hydroxyapatite from mitochondria of a NSC34 cell line expressing human SOD1G93A, a suitable ALS motor neuron model, were analyzed by tryptic and chymotryptic proteolysis and UHPLC/High-Resolution ESI-MS/MS. We found selective deamidations of asparagine and glutamine of VDAC1 in ALS-related NSC34-SOD1G93A cells but not in NSC34-SOD1WT or NSC34 cells. In addition, we identified differences in the over-oxidation of methionine and cysteines between VDAC1 purified from ALS model or non-ALS NSC34 cells. The specific range of PTMs identified exclusively in VDAC1 from NSC34-SOD1G93A cells but not from NSC34 control lines, suggests the appearance of important changes to the structure of the VDAC1 channel and therefore to the bioenergetics metabolism of ALS motor neurons. Data are available via ProteomeXchange with identifier .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/antiox9121218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761621PMC
December 2020

Identification of New Antimicrobial Peptides from Mediterranean Medical Plant (Steinh.) Speta.

Antibiotics (Basel) 2020 Oct 28;9(11). Epub 2020 Oct 28.

Istituto Zooprofilattico Sperimentale della Sicilia "A. Mirri", 90129 Palermo, Italy.

The present work was designed to identify and characterize novel antimicrobial peptides (AMPs) from (Steinh.) Speta, previously named , is a Mediterranean plant, well-known for its biological properties in traditional medicine. Polypeptide-enriched extracts from different parts of the plant (roots, leaves and bulb), never studied before, were tested against two relevant pathogens, and . With the aim of identifying novel natural AMPs, peptide fraction displaying antimicrobial activity (the bulb) that showed minimum inhibitory concentration (MICs) equal to 30 µg/mL against the above mentioned strains, was analysed by high-resolution mass spectrometry and database search. Seventeen peptides, related to seven proteins present in the investigated database, were described. Furthermore, we focused on three peptides, which due to their net positive charge, have a better chance to be AMPs and they were investigated by molecular modelling approaches, in order to shed light on the solution properties of their equilibrium structures. Some of new detected peptides could represent a good platform for the development of new antimicrobials in the fight against antibiotic resistance phenomenon.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/antibiotics9110747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7694139PMC
October 2020

Mass Spectrometry and H-NMR Study of (Quebracho) Tannins as a Source of Hypoglycemic and Antioxidant Principles.

Molecules 2020 Jul 17;25(14). Epub 2020 Jul 17.

Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy.

The ethyl acetate extract of the commercial tannin Tan'Activ QS-SOL (from wood), employed for the production of red wine, was subjected to chromatography on Sephadex LH-20, providing nine fractions (A-1-A-9), which were estimated for total phenols content (GAE), antioxidant activity (DPPH, ORAC), and hypoglycemic activity (α-glucosidase and α-amylase inhibition). All the fractions were analyzed by means of HPLC/ESI-MS/MS and H-NMR to identify the principal active constituents. Fractions A-1 and A-3 showed the highest antioxidant activity and gallic acid (), pyrogallol (), eriodictyol (), catechin (), and taxifolin () were identified as the major constituents. The highest α-glucosidase and α-amylase inhibitory activity was observed in fractions A-7-A-9 containing condensed (, , , , , and ) hydrolysable tannins ( and ) as well as esters of quinic acid with different units of gallic acid (, , , , and ). This last class of gallic acid esters are here reported for the first time as α-glucosidase and α-amylase inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/molecules25143257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7397293PMC
July 2020

Defective proteasome biogenesis into skin fibroblasts isolated from Rett syndrome subjects with MeCP2 non-sense mutations.

Biochim Biophys Acta Mol Basis Dis 2020 07 8;1866(7):165793. Epub 2020 Apr 8.

Dept of Clinical Sciences and Translational Medicine, University of Rome Tor Vergata, Rome, Italy. Electronic address:

Rett Syndrome (RTT) is a rare X-linked neurodevelopmental disorder which affects about 1: 10000 live births. In >95% of subjects RTT is caused by a mutation in Methyl-CpG binding protein-2 (MECP2) gene, which encodes for a transcription regulator with pleiotropic genetic/epigenetic activities. The molecular mechanisms underscoring the phenotypic alteration of RTT are largely unknown and this has impaired the development of therapeutic approaches to alleviate signs and symptoms during disease progression. A defective proteasome biogenesis into two skin primary fibroblasts isolated from RTT subjects harbouring non-sense (early-truncating) MeCP2 mutations (i.e., R190fs and R255X) is herewith reported. Proteasome is the proteolytic machinery of Ubiquitin Proteasome System (UPS), a pathway of overwhelming relevance for post-mitotic cells metabolism. Molecular, transcription and proteomic analyses indicate that MeCP2 mutations down-regulate the expression of one proteasome subunit, α7, and of two chaperones, PAC1 and PAC2, which bind each other in the earliest step of proteasome biogenesis. Furthermore, this molecular alteration recapitulates in neuron-like SH-SY5Y cells upon silencing of MeCP2 expression, envisaging a general significance of this transcription regulator in proteasome biogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbadis.2020.165793DOI Listing
July 2020

A High Resolution Mass Spectrometry Study Reveals the Potential of Disulfide Formation in Human Mitochondrial Voltage-Dependent Anion Selective Channel Isoforms (hVDACs).

Int J Mol Sci 2020 Feb 21;21(4). Epub 2020 Feb 21.

Department of Chemical Sciences, Organic Mass Spectrometry Laboratory, University of Catania, Viale A. Doria 6, 95125 Catania, Italy.

The voltage-dependent anion-selective channels (VDACs), which are also known as eukaryotic porins, are pore-forming proteins, which allow for the passage of ions and small molecules across the outer mitochondrial membrane (OMM). They are involved in complex interactions that regulate organelle and cellular metabolism. We have recently reported the post-translational modifications (PTMs) of the three VDAC isoforms purified from rat liver mitochondria (rVDACs), showing, for the first time, the over-oxidation of the cysteine residues as an exclusive feature of VDACs. Noteworthy, this peculiar PTM is not detectable in other integral membrane mitochondrial proteins, as defined by their elution at low salt concentration by a hydroxyapatite column. In this study, the association of tryptic and chymotryptic proteolysis with UHPLC/High Resolution nESI-MS/MS, allowed for us to extend the investigation to the human VDACs. The over-oxidation of the cysteine residues, essentially irreversible in cell conditions, was as also certained in VDAC isoforms from human cells. In human VDAC2 and 3 isoforms the permanently reduced state of a cluster of close cysteines indicates the possibility that disulfide bridges are formed in the proteins. Importantly, the detailed oxidative PTMs that are found in human VDACs confirm and sustain our previous findings in rat tissues, claiming for a predictable characterization that has to be conveyed in the functional role of VDAC proteins within the cell. Data are available via ProteomeXchange with identifier PXD017482.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms21041468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073118PMC
February 2020

Dataset of the metabolic and CM-like protein fractions in old and modern wheat Italian genotypes.

Data Brief 2019 Dec 1;27:104730. Epub 2019 Nov 1.

Laboratory of Organic Mass Spectrometry, Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125, Catania, Italy.

The present work reports the first comprehensive proteomic profiling and qualitative comparison of metabolic and Chloroform-Methanol (CM)-like protein fractions extracted from mature kernels of two old Sicilian durum wheat landraces, and , and , an improved durum wheat variety widespread in Italy and other Mediterranean countries and chosen as representative of the most widely commercial cultivars. The data are discussed in the related research article "Qualitative proteomic comparison of metabolic and CM-like protein fractions in old and modern wheat Italian genotypes by a shotgun approach" [1]. The results of this work could be used for investigations to understand the relationship between protein profiles of old and modern wheat genotypes and their potential benefits for human consumption.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.dib.2019.104730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6859223PMC
December 2019

Qualitative proteomic comparison of metabolic and CM-like protein fractions in old and modern wheat Italian genotypes by a shotgun approach.

J Proteomics 2020 01 16;211:103530. Epub 2019 Oct 16.

Laboratory of Organic Mass Spectrometry, Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy.

The close relationship between diet and health is generally recognized and the growing wellness and consciousness, especially in developed countries, have led to increasing interest for old wheat genotypes, based on perceived health benefits. Although nutritional comparison between old and modern wheat varieties is still controversial, it is generally accepted that old wheat genotypes remained unchanged over the last hundred years. By contrast, modern wheat genotypes are derived by modification of old wheats during the so-called "Green-Revolution" in the second half of the 20th century focusing on obtaining properties in terms of higher grain yield. The present work reports the first comprehensive proteomic profiling and qualitative comparison at the molecular level of metabolic and Chloroform-Methanol (CM)-like protein fractions extracted from mature kernels of two old Sicilian durum wheat landraces, Russello and Timilia Reste Bianche, and Simeto, an improved durum wheat variety widespread in Italy and other Mediterranean countries and chosen as representative of the most widely commercial cultivars. The results obtained reveal that metabolic and CM-like protein fractions of old and modern genotypes present remarkably high similarity with only minor differences. This leads to the conclusion that from a food and nutritional perspective there is a substantial equivalence of the protein composition of the old and modern cultivars. Data are available via ProteomeXchange with identifier PXD014449. BIOLOGICAL SIGNIFICANCE: In recent years consumers have shown growing interest in the old wheat genotypes, which are generally perceived more "natural" and healthier than modern ones. However, comparison of nutritional value for modern and old wheat varieties is still controversial suggesting further studies. In particular proteome analysis of old and modern wheat genotypes is currently ongoing with particular focus on gluten proteins, whereas the metabolic protein fraction has not yet been investigated. In the present study, we conducted a comprehensive proteomic profile and qualitative comparison at the molecular level of metabolic and Chloroform-Methanol (CM)-like protein fractions of the old Sicilian landraces Russello and Timilia Reste Bianche and the modern cultivar Simeto by applying a shotgun approach. The results reveal that the metabolic and CM-like protein fractions of old and modern genotypes are remarkably similar with only minor differences, leading to the conclusion that from a food and nutritional perspective there is a substantial equivalence of these cultivars. These results may contribute to improved understanding of the relationship between protein profiles of old wheat genotypes and their potential benefits for human consumption.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jprot.2019.103530DOI Listing
January 2020

Sequential Fractionation Strategy Identifies Three Missing Proteins in the Mitochondrial Proteome of Commonly Used Cell Lines.

J Proteome Res 2018 12 5;17(12):4307-4314. Epub 2018 Oct 5.

Institute of Biochemistry and Clinical Biochemistry , Università Cattolica del Sacro Cuore , L.go F. Vito 1 , 00168 Rome , Italy.

Mitochondria are undeniably the cell powerhouse, directly affecting cell survival and fate. Growing evidence suggest that mitochondrial protein repertoire affects metabolic activity and plays an important role in determining cell proliferation/differentiation or quiescence shift. Consequently, the bioenergetic status of a cell is associated with the quality and abundance of the mitochondrial populations and proteomes. Mitochondrial morphology changes in the development of different cellular functions associated with metabolic switches. It is therefore reasonable to speculate that different cell lines do contain different mitochondrial-associated proteins, and the investigation of these pools may well represent a source for mining missing proteins (MPs). A very effective approach to increase the number of IDs through mass spectrometry consists of reducing the complexity of the biological samples by fractionation. The present study aims at investigating the mitochondrial proteome of five phenotypically different cell lines, possibly expressing some of the MPs, through an enrichment-fractionation approach at the organelle and protein level. We demonstrate a substantial increase in the proteome coverage, which, in turn, increases the likelihood of detecting low abundant proteins, often falling in the category of MPs, and resulting, for the present study, in the identification of METTL12, FAM163A, and RGS13. All MS data have been deposited to the MassIVE data repository ( https://massive.ucsd.edu ) with the data set identifier MSV000082409 and PXD010446.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jproteome.8b00422DOI Listing
December 2018

Proteomic Analyses on an Ancient Egyptian Cheese and Biomolecular Evidence of Brucellosis.

Anal Chem 2018 08 30;90(16):9673-9676. Epub 2018 Jul 30.

Department of Chemical Sciences , University of Catania , Viale A. Doria 6-I , 95125 Catania , Italy.

The material analyzed in this study is probably the most ancient archeological solid residue of cheese ever found to date. The sample was collected during the Saqqara Cairo University excavations in the tomb of Ptahmes dated to XIX dynasty ( El-Aguizy, O. Bulletin de l'Institut Française d'Archéologie Orientale (BIFAO) 2010 , 110 , 13 - 34 (ref (1) ); Staring, N. Bulletin de Institut Français d'Archéologie Orientale (BIFAO) 2015 , 114 , 455 - 518 (ref (2) )). Our biomolecular proteomic characterization of this archeological sample shows that the constituting material was a dairy product obtained by mixing sheep/goat and cow milk. The interactions for thousands of years with the strong alkaline environment of the incorporating soil rich in sodium carbonate and the desertic conditions did not prevent the identification of specific peptide markers which showed high stability under these stressing conditions. Moreover, the presence of Brucella melitensis has been attested by specific peptide providing a reasonable direct biomolecular evidence of the presence of this infection in the Ramesside period for which only indirect paleopathological evidence has been so far provided ( Pappas, G.; Papadimitriou P. Int. J. Antimicrob. Agents 2007 , 30 , 29 - 31 (ref (3) ); Bourke, J. B. Medical History 1971 , 15 ( 4 ), 363 - 375 (ref (4) )). Finally, it is worth noting that, although proteomic approaches are successfully and regularly used to characterize modern biological samples ( D'Ambrosio, C.; Arena, S.; Salzano, A. M.; Renzone, G.; Ledda, L.; and Scaloni, A. Proteomics 2008 8 , 3657 - 3666 (ref (5) ), their application in ancient materials is still at an early stage of progress, only few results being reported about ancient food samples ( Yang, Y.; Shevchenko, A.; Knaust, A.; Abuduresule, I.; Li, W.; Hu, X.; Wang, C.; Shevchenko, A. J. Archaeol. Sci. 2014 , 45 , 178 - 186 (ref (6) ). In the absence of previous relevant evidence of cheese production and/or use, this study, undoubtedly has a clear added value in different fields of knowledge ranging from archaeometry, anthropology, archeology, medicine history to the forensic sciences.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.analchem.8b02535DOI Listing
August 2018

Post-translational modifications of VDAC1 and VDAC2 cysteines from rat liver mitochondria.

Biochim Biophys Acta Bioenerg 2018 09 8;1859(9):806-816. Epub 2018 Jun 8.

National Institute for Biomembranes and Biosystems, Section of Catania, Viale A. Doria 6, 95125 Catania, Italy; Department of Biomedicine and Biotechnology, Section of Biology and Genetics, Viale A. Doria 6, 95125, Catania, Italy. Electronic address:

VDACs three isoforms (VDAC1, VDAC2, VDAC3) are integral proteins of the outer mitochondrial membrane whose primary function is to permit the communication and exchange of molecules related to the mitochondrial functions. We have recently reported about the peculiar over-oxidation of VDAC3 cysteines. In this work we have extended our analysis, performed by tryptic and chymotryptic proteolysis and UHPLC/High Resolution ESI-MS/MS, to the other two isoforms VDAC1 and VDAC2 from rat liver mitochondria, and we have been able to find also in these proteins over-oxidation of cysteines. Further PTM of cysteines as succination has been found, while the presence of selenocysteine was not detected. Unfortunately, a short sequence stretch containing one genetically encoded cysteine was not covered both in VDAC2 and in VDAC3, raising the suspect that more, unknown modifications of these proteins exist. Interestingly, cysteine over-oxidation appears to be an exclusive feature of VDACs, since it is not present in other transmembrane mitochondrial proteins eluted by hydroxyapatite. The assignment of a functional role to these modifications of VDACs will be a further step towards the full understanding of the roles of these proteins in the cell.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbabio.2018.06.007DOI Listing
September 2018

Polymorphism at donkey β-lactoglobulin II locus: identification and characterization of a new genetic variant with a very low expression.

Amino Acids 2018 Jun 23;50(6):735-746. Epub 2018 Mar 23.

Animal Production Section, Department of Agriculture, Food and Environment, University of Catania, Via Valdisavoia 5, 95123, Catania, Italy.

In the last years, donkey milk had evidenced a renewed interest as a potential functional food and a breast milk substitute. In this light, the study of the protein composition assumes an important role. In particular, β-lactoglobulin (β-LG), which is considered as one of the main allergenic milk protein, in donkey species consists of two molecular forms, namely β-LG I and β-LG II. In the present research, a genetic analysis coupled with a proteomic approach showed the presence of a new allele, here named F, which is apparently associated with a null or a severely reduced expression of β-LG II protein. The new β-LG II F genetic variant shows a theoretical average mass (M) of 18,310.64 Da, a value practically corresponding with that of the variant D (∆ < 0.07 Da), but differs from β-LG II D for two amino acid substitutions: Thr (variant F) → Ala (variant D) and Thr (variant F) → Met (variant D). Proteomic investigation of the whey protein fraction of an individual milk sample, homozygous FF at β-LG II locus, allowed to identify, as very minor component, the new β-LG II F genetic variant. By MS/MS analysis of enzymatic digests, the sequence of the β-LG II F was characterized, and the predicted genomic data confirmed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00726-018-2555-1DOI Listing
June 2018

Toward the Standardization of Mitochondrial Proteomics: The Italian Mitochondrial Human Proteome Project Initiative.

J Proteome Res 2017 12 13;16(12):4319-4329. Epub 2017 Sep 13.

Department of Science and High Technology, Università degli Studi dell'Insubria , Busto Arsizio I-21052, Italy.

The Mitochondrial Human Proteome Project aims at understanding the function of the mitochondrial proteome and its crosstalk with the proteome of other organelles. Being able to choose a suitable and validated enrichment protocol of functional mitochondria, based on the specific needs of the downstream proteomics analysis, would greatly help the researchers in the field. Mitochondrial fractions from ten model cell lines were prepared using three enrichment protocols and analyzed on seven different LC-MS/MS platforms. All data were processed using neXtProt as reference database. The data are available for the Human Proteome Project purposes through the ProteomeXchange Consortium with the identifier PXD007053. The processed data sets were analyzed using a suite of R routines to perform a statistical analysis and to retrieve subcellular and submitochondrial localizations. Although the overall number of identified total and mitochondrial proteins was not significantly dependent on the enrichment protocol, specific line to line differences were observed. Moreover, the protein lists were mapped to a network representing the functional mitochondrial proteome, encompassing mitochondrial proteins and their first interactors. More than 80% of the identified proteins resulted in nodes of this network but with a different ability in coisolating mitochondria-associated structures for each enrichment protocol/cell line pair.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jproteome.7b00350DOI Listing
December 2017

Proteins and bioactive peptides from donkey milk: The molecular basis for its reduced allergenic properties.

Food Res Int 2017 09 4;99(Pt 1):41-57. Epub 2017 Jul 4.

Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy.

The legendary therapeutics properties of donkey milk have recently been supported by many clinical trials who have clearly demonstrated that, even if with adequate lipid integration, it may represent a valid natural substitute of cow milk for feeding allergic children. During the last decade many investigations by MS-based methods have been performed in order to obtain a better knowledge of donkey milk proteins. The knowledge about the primary structure of donkey milk proteins now may provide the basis for a more accurate comprehension of its potential benefits for human nutrition. In this aspect, experimental data today available clearly demonstrate that donkey milk proteins (especially casein components) are more closely related with the human homologues rather than cow counterparts. Moreover, the low allergenic properties of donkey milk with respect to cow one seem to be related to the low total protein content, the low ratio of caseins to whey fraction, and finally to the presence in almost all bovine IgE-binding linear epitopes of multiple amino acid differences with respect to the corresponding regions of donkey milk counterparts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.foodres.2017.07.002DOI Listing
September 2017

High resolution mass spectrometry characterization of the oxidation pattern of methionine and cysteine residues in rat liver mitochondria voltage-dependent anion selective channel 3 (VDAC3).

Biochim Biophys Acta Biomembr 2017 03 16;1859(3):301-311. Epub 2016 Dec 16.

Department of Chemical Sciences, Organic Mass Spectrometry Laboratory, University of Catania, Viale A. Doria 6, 95125 Catania, Italy. Electronic address:

Voltage-dependent anion selective channels (VDACs) are integral membrane proteins found in the mitochondrial outer membrane. In comparison with the most abundant isoform VDAC1, there is little knowledge about the functional role of VDAC3. Unlikely VDAC1, cysteine residues are particularly abundant in VDAC3. Since the mitochondrial intermembrane space (IMS) has an oxidative potential we questioned whether the redox state of VDAC3 can be modified. By means of SDS-PAGE separation, tryptic and chymotryptic proteolysis and UHPLC/High Resolution ESI-MS/MS analysis we investigated the oxidation state of cysteine and methionine residues of rat liver VDAC3. Our results demonstrate that the mitochondrial VDAC3, in physiological state, contains methionines oxidized to methionine sulfoxide. Furthermore, cysteine residues 36, 65, and 165 are oxidized to a remarkable extend to sulfonic acid. Cysteines 2 and 8 are observed exclusively in the carboxyamidomethylated form. Cys is detected exclusively in the oxidized form of sulfonic acid, whereas the oxidation state of Cys could not be determined because peptides containing this residue were not detected. Control experiments ruled out the possibility that over-oxidation of cysteines might be due to artefactual reasons. The peculiar behavior of Met and Cys residues of VDAC3 may be related with the accessibility of the protein to a strongly oxidizing environment and may be connected with the regulation of the activity of this trans-membrane pore protein.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbamem.2016.12.003DOI Listing
March 2017

Polyphemus, Odysseus and the ovine milk proteome.

J Proteomics 2017 01 23;152:58-74. Epub 2016 Oct 23.

Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy.

In the last years the amount of ovine milk production, mainly used to formulate a wide range of different and exclusive dairy products often categorized as gourmet food, has been progressively increasing. Taking also into account that sheep milk (SM) also appears to be potentially less allergenic than cow's one, an in-depth information about its protein composition is essential to improve the comprehension of its potential benefits for human consumption. The present work reports the results of an in-depth characterization of SM whey proteome, carried out by coupling the CPLL technology with SDS-PAGE and high resolution UPLC-nESI MS/MS analysis. This approach allowed the identification of 718 different protein components, 644 of which are from unique genes. Particularly, this identification has expanded literature data about sheep whey proteome by 193 novel proteins previously undetected, many of which are involved in the defence/immunity mechanisms or in the nutrient delivery system. A comparative analysis of SM proteome known to date with cow's milk proteome, evidenced that while about 29% of SM proteins are also present in CM, 71% of the identified components appear to be unique of SM proteome and include a heterogeneous group of components which seem to have health-promoting benefits. The data have been deposited to the ProteomeXchange with identifier .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jprot.2016.10.007DOI Listing
January 2017

Site-specific glycosylation of donkey milk lactoferrin investigated by high-resolution mass spectrometry.

Amino Acids 2016 12 22;48(12):2799-2808. Epub 2016 Aug 22.

Protein Research Group, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230, Odense M, Denmark.

A comprehensive monosaccharide composition of the N-glycans of donkey milk lactoferrin, isolated by ion exchange chromatography from an individual milk sample, was obtained by means of chymotryptic digestion, TiO and HILIC enrichment, reversed-phase high-performance liquid chromatography, electrospray mass spectrometry, and high collision dissociation fragmentation. The results obtained allowed identifying 26 different glycan structures, including high mannose, complex and hybrid N-glycans, linked to the protein backbone via an amide bond to asparagine residues located at the positions 137, 281 and 476. Altogether, the N-glycan structures determined revealed that most of the N-glycans identified in donkey milk lactoferrin are neutral complex/hybrid. Indeed, 10 neutral non-fucosylated complex/hybrid N-glycans and 4 neutral fucosylated complex/hybrid N-glycans were found. In addition, two high mannose N-glycans, four sialylated fucosylated complex N-glycans and six sialylated non-fucosylated complex N-glycans, one of which containing N-glycolylneuraminic acid (Neu5Gc), were found. A comparison of the monosaccharide composition of the N-glycans of donkey milk lactoferrin with respect to that of human, bovine and goat milk lactoferrin is reported. Data are available via ProteomeXchange with identifier PXD004289.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00726-016-2315-zDOI Listing
December 2016

α-Glucosidase inhibition and antioxidant activity of an oenological commercial tannin. Extraction, fractionation and analysis by HPLC/ESI-MS/MS and (1)H NMR.

Food Chem 2017 Jan 25;215:50-60. Epub 2016 Jul 25.

Dipartimento di Scienze Chimiche, Università degli Studi di Catania, V.le A. Doria 6, 95125 Catania, Italy. Electronic address:

Two batches of the oenological tannin Tan'Activ R, (toasted oak wood - Quercus robur), were extracted with ethanol. A fractionation on XAD-16 afforded four fractions for each extract. Extracts and fractions were evaluated for antioxidant activity (DPPH), polyphenol content (GAE) and yeast α-glucosidase inhibitory activity. Comparable results were obtained for both columns, fractions X1B and X2B showing the highest antioxidant activity. Fractions X1C and X2C notably inhibited α-glucosidase, with IC50=9.89 and 8.05μg/mL, respectively. Fractions were subjected to HPLC/ESI-MS/MS and (1)H NMR analysis. The main phenolic constituents of both X1B and X2B were a monogalloylglucose isomer (1), a HHDP-glucose isomer (2), castalin (3) gallic acid (4), vescalagin (5), and grandinin (or its isomer roburin E, 6). X1C and X2C showed a complex composition, including non-phenolic constituents. Fractionation of X2C gave a subfraction, with enhanced α-glucosidase inhibitory activity (IC50=6.15μg/mL), with castalagin (7) as the main constituent.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.foodchem.2016.07.136DOI Listing
January 2017

Sequence characterization and glycosylation sites identification of donkey milk lactoferrin by multiple enzyme digestions and mass spectrometry.

Amino Acids 2016 07 28;48(7):1569-80. Epub 2016 Mar 28.

Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230, Odense, Denmark.

Lactoferrin, a protein showing an array of biochemical properties, including immuno-modulation, iron-binding ability, as well as antioxidant, antibacterial and antiviral activities, but which may also represent a potential milk allergen, was isolated from donkey milk by ion exchange chromatography. The characterization of its primary structure, by means of enzymatic digestions, SPITC derivatization of tryptic digest, reversed-phase high performance liquid chromatography, electrospray and matrix-assisted laser desorption/ionization mass spectrometry, is reported. Our results allowed the almost complete characterization of donkey lactoferrin sequence, that, at least for the covered sequence, differs from the horse genomic deduced sequence (UniProtKB Acc. Nr. O77811) by five point substitutions located at positions 91 (Arg → His), 328 (Thr → Ile/Leu), 466 (Ala → Gly), 642 (Asn → Ser) and 668 (Ser → Ala). Analysis of the glycosylated protein showed that glycans in donkey lactoferrin are linked to the protein backbone via an amide bond to asparagine residues located at the positions 137, 281 and 476.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00726-016-2209-0DOI Listing
July 2016

Zeus, Aesculapius, Amalthea and the proteome of goat milk.

J Proteomics 2015 Oct 17;128:69-82. Epub 2015 Jul 17.

Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy.

The goat whey proteome has been explored in depth via capture with combinatorial peptide ligand libraries (CPLLs) at three different pH values. A total of 452 unique species have been tabulated, a proteome discovery so far unmatched in any single other investigation of milk from any mammalian species. This massive discovery is probably related to: i) the extraordinary load of proteins onto the CPLL beads (i.e. 2 g for each different pH captures) vs. barely 100 μL of beads; ii) the high resolution/high mass accuracy of mass spectral data; and iii) the use of two complementary tools, Mascot and PEAKS, each one contributing to a set of unique protein IDs. Due to the relative paucity of available protein annotations for goat, only 10% of the identified proteins belong to the capra, whereas 52% are specific of sheep and 37% are homologous to that of bovine milk. This work reports the largest description so far of the goat milk proteome, which has been compared with cow's milk proteome and would thus help to understand the importance of low-abundance proteins with respect to the unique biological properties of this nutrient.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jprot.2015.07.009DOI Listing
October 2015

Protein profile of exhaled breath condensate determined by high resolution mass spectrometry.

J Pharm Biomed Anal 2015 Feb 5;105:134-149. Epub 2014 Dec 5.

Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy.

A method based on liquid chromatography/high resolution tandem mass spectrometry coupled with electrophoretic separation, for determination and relative quantification of the protein composition of exhaled breath condensate (EBC), was developed. Application of the procedure to a sample of EBC, pooled from nine healthy subjects, resulted in the identification of 167 unique gene products, 113 of which not previously reported in EBC samples. The abundance of the protein identified was estimated by means of the exponentially modified protein abundance index protocol (emPAI). Cytokeratins were by far the most abundant proteins in EBC samples. Many of the identified proteins were associated with multiple cellular location with cytoplasm constituting the largest group. Cytosol, nucleus, membrane, cytoskeleton and extracellular were other abundantly represented locations. No amylase was detected, suggesting the absence of saliva protein contamination. The profile obtained represents the most comprehensive protein characterization of EBC so far reported and demonstrates that this approach provides a powerful tool for investigating the protein profile of EBC samples. Compared with analogous investigations, this study also shows that the protein profile of EBC is strongly affected by the sampling method adopted.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jpba.2014.11.050DOI Listing
February 2015

Mass spectrometry in food proteomics: a tutorial.

J Mass Spectrom 2014 Sep;49(9):768-84

Department of Chemical Sciences, University of Catania, Viale A. Doria, 6, I-95125, Catania, Italy.

In the last decades, the continuous and rapid evolution of proteomic approaches has provided an efficient platform for the characterization of food-derived proteins. Particularly, the impressive increasing in performance and versatility of the MS instrumentation has contributed to the development of new analytical strategies for proteins, evidencing how MS arguably represents an indispensable tool in food proteomics. Investigation of protein composition in foodstuffs is helpful for understanding the relationship between the protein content and the nutritional and technological properties of foods, the production of methods for food traceability, the assessment of food quality and safety, including the detection of allergens and microbial contaminants in foods, or even the characterization of genetically modified products. Given the high variety of the food-derived proteins and considering their differences in chemical and physical properties, a single proteomic strategy for all purposes does not exist. Rather, proteomic approaches need to be adapted to each analytical problem, and development of new strategies is necessary in order to obtain always the best results. In this tutorial, the most relevant aspects of MS-based methodologies in food proteomics will be examined, and their advantages and drawbacks will be discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jms.3374DOI Listing
September 2014

Root protein profiles of two citrus rootstocks grown under iron sufficiency/deficiency conditions.

Eur J Mass Spectrom (Chichester) 2013 ;19(4):305-24

Dipartimento di Scienze Chimiche, Università di Catania, Viate A. Doria 6, 95125 Catania, Italy.

Two citrus rootstocks, one sensitive to iron deficiency (Swingle Citrumelo (SCO)) and the other tolerant (Carrizo Citrange, (CC)), were studied to characterize variation in their root protein profile induced by iron-deficient conditions. Plants of both rootstocks were grown in two different soils, one volcanic (v) and the other calcareous (c), containing 0% and 10% active Lime, respectively. To evaluate the effects of the calcareous soil on the protein accumulation of both rootstocks, the root protein profiles (SCc vs. SCv and CCc vs. CCv) were characterized by two-dimensional gel electrophoresis, thus obtaining, for the first time, a reference map of this previously uncharacterized proteome. A total of 219 spots, significantly changed in abundance, were analyzed by high-performance Liquid chromatography nano electrospray ionization tandem mass spectrometry. The identified proteins were classified according to their putative function and known biosynthetic pathways. Principal component analysis, comparing the four sets of data, indicated that each group clustered together with low variance and that CCv and CCc data sets were well differentiated, whereas SCv and SCc were similar.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1255/ejms.1230DOI Listing
April 2014

Involvement of alanine racemase in germination of Bacillus cereus spores lacking an intact exosporium.

Arch Microbiol 2014 Feb 18;196(2):79-85. Epub 2013 Dec 18.

Department of Food Science, University of Udine, Via Sondrio 2/A, 33100, Udine, Italy.

The L-alanine mediated germination of food isolated Bacillus cereus DSA 1 spores, which lacked an intact exosporium, increased in the presence of D-cycloserine (DCS), which is an alanine racemase (Alr) inhibitor, reflecting the activity of the Alr enzyme, capable of converting L-alanine to the germination inhibitor D-alanine. Proteomic analysis of the alkaline extracts of the spore proteins, which include exosporium and coat proteins, confirmed that Alr was present in the B. cereus DSA 1 spores and matched to that encoded by B. cereus ATCC 14579, whose spore germination was strongly affected by the block of conversion of L- to D-alanine. Unlike ATCC 14579 spores, L-alanine germination of B. cereus DSA 1 spores was not affected by the preincubation with DCS, suggesting a lack of restriction in the reactant accessibility.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00203-013-0946-yDOI Listing
February 2014

Immune mediators of sea-cucumber Holothuria tubulosa (Echinodermata) as source of novel antimicrobial and anti-staphylococcal biofilm agents.

AMB Express 2013 Jun 24;3(1):35. Epub 2013 Jun 24.

Department STEBICEF, Università degli Studi di Palermo, Via Archirafi, 32, Palermo, 90123, Italy.

The present study aims to investigate coelomocytes, immune mediators cells in the echinoderm Holothuria tubulosa, as an unusual source of antimicrobial and antibiofilm agents. The activity of the 5kDa peptide fraction of the cytosol from H. tubulosa coelomocytes (5-HCC) was tested against a reference group of Gram-negative and Gram-positive human pathogens. Minimal inhibitory concentrations (MICs) ranging from 125 to 500 mg/ml were determined against tested strains. The observed biological activity of 5-HCC could be due to two novel peptides, identified by capillary RP-HPLC/nESI-MS/MS, which present the common chemical-physical characteristics of antimicrobial peptides. Such peptides were chemically synthesized and their antimicrobial activity was tested. The synthetic peptides showed broad-spectrum activity at 12.5 mg/ml against the majority of the tested Gram-positive and Gram-negative strains, and they were also able to inhibit biofilm formation in a significant percentage at a concentration of 3.1 mg/ml against staphylococcal and Pseudomonas aeruginosa strains.The immune mediators in H. tubulosa are a source of novel antimicrobial peptides for the development of new agents against biofilm bacterial communities that are often intrinsically resistant to conventional antibiotics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/2191-0855-3-35DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3702452PMC
June 2013

MALDI-TOF mass spectrometry for the monitoring of she-donkey's milk contamination or adulteration.

J Mass Spectrom 2013 Feb;48(2):148-53

Department of Chemical Science, University of Catania, Viale A. Doria 6, 95125, Catania, Italy.

Donkey's milk (DM), representing a safe and alternative food in both IgE-mediated and non-IgE-mediated cow's milk protein allergy, can be categorized as precious pharma-food. Moreover, an economically relevant interest for the use of DM in cosmetology is also developing. The detection of adulterations and contaminations of DM is a matter of fundamental importance from both an economic and allergenic standpoint, and, to this aim, fast and efficient analytical approaches to assess the authenticity of this precious nutrient are desirable. Here, a rapid matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS)-based method aimed to the detection of bovine or caprine milk in raw DM is reported. The presence of the extraneous milks was revealed by monitoring the protein profiles of the most abundant whey proteins, α-lactalbumin (α-LA) and β-lactoglobulin, used as molecular markers. The possibility of obtaining a quantitative analysis of the level of cow or goat milk in DM based on the MALDI-TOF peak areas of α-LAs was also explored. The results showed that the experimental quantitative values were in good agreement with the real composition of each mixture. As pretreatment of the milk samples is not required, and owing to the speed and the high sensitivity of MALDI-MS, the protocol here reported could represent a reliable method for routine analyses aimed to assess the absence of contamination in raw fresh DM samples.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jms.3138DOI Listing
February 2013

MS-based characterization of α(s2)-casein isoforms in donkey's milk.

J Mass Spectrom 2012 Sep;47(9):1150-9

Department of Chemical Sciences, University of Catania, Viale A. Doria 6, I-95125, Catania, Italy.

The primary structure of four α(s2)-casein (CN) isoforms, present as minor components in the dephosphorylated CN fraction of a milk sample collected in Eastern Sicily from an individual donkey belonging to the Ragusano breed at middle lactation stage, was determined, using the known donkey's α(s2)-CN (GenBank Acc. No. CAV00691; M(r) 26,028 Da) as reference. Proteins, with experimentally measured M(r) of 25,429, 21,939, 25,203 and 21,713 Da, were isolated by the combined use of reversed-phase high-performance liquid chromatography (RP-HPLC) and two-dimensional polyacrylamide gel electrophoresis. The major spot of each gel, corresponding to a single protein, was digested by trypsin, α-chymotrypsin and endoproteinase Glu-C. The resulting peptide mixtures were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and capillary RP-HPLC/nano-electrospray ionization tandem mass spectrometry, and the data obtained were used for the sequence determination. The isoforms are produced from differential splicing events involving exons 4, 5 and 6 and parts of the exon 17.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jms.3031DOI Listing
September 2012

Mass spectrometry in the proteome analysis of mature cereal kernels.

Mass Spectrom Rev 2012 Jul-Aug;31(4):448-65. Epub 2011 Aug 22.

Dipartimento di Scienze Chimiche, Università degli Studi di Catania, Italy.

In the last decade, the improved performance and versatility of the mass spectrometers together with the increasing availability of gene and genomic sequence database, led the mass spectrometry to become an indispensable tool for either protein and proteome analyses in cereals. Mass spectrometric works on prolamins have rapidly evolved from the determination of the molecular masses of proteins to the proteomic approaches aimed to a large-scale protein identification and study of functional and regulatory aspects of proteins. Mass spectrometry coupled with electrophoresis, chromatographic methods, and bioinformatics tools is currently making significant contributions to a better knowledge of the composition and structure of the cereal proteins and their structure-function relationships. Results obtained using mass spectrometry, including characterization of prolamins, investigation of the gluten toxicity for coeliac patients, identification of proteins responsible of cereal allergies, determination of the protein pattern and its modification under environmental or stress effects, investigation of genetically modified varieties by proteomic approaches, are summarized here, to illustrate current trends, analytical troubles and challenges, and suggest possible future perspectives.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/mas.20347DOI Listing
October 2012

Applications of liquid chromatography-mass spectrometry for food analysis.

J Chromatogr A 2012 Oct 17;1259:74-85. Epub 2012 Apr 17.

Department of Molecular and Biomolecular Sciences and Technologies (STEMBIO), University of Palermo, via Archirafi 32, 90123 Palermo, Italy.

HPLC-MS applications in the agrifood sector are among the fastest developing fields in science and industry. The present tutorial mini-review briefly describes this analytical methodology: HPLC, UHPLC, nano-HPLC on one hand, mass spectrometry (MS) and tandem mass spectrometry (MS/MS) on the other hand. Analytical results are grouped together based on the type of chemicals analyzed (lipids, carbohydrates, glycoproteins, vitamins, flavonoids, mycotoxins, pesticides, allergens and food additives). Results are also shown for various types of food (ham, cheese, milk, cereals, olive oil and wines). Although it is not an exhaustive list, it illustrates the main current directions of applications. Finally, one of the most important features, the characterization of food quality (including problems of authentication and adulteration) is discussed, together with a future outlook on future directions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chroma.2012.04.023DOI Listing
October 2012