Publications by authors named "Salvatore Foti"

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

A New Approach for Impedance Tracking of Piezoelectric Vibration Energy Harvesters Based on a Zeta Converter.

Sensors (Basel) 2020 Oct 16;20(20). Epub 2020 Oct 16.

Department of Engineering, University of Messina, 98166 Messina, Italy.

Piezoelectric energy harvesters (PEHs) are a reduced, but fundamental, source of power for embedded, remote, and no-grid connected electrical systems. Some key limits, such as low power density, poor conversion efficiency, high internal impedance, and AC output, can be partially overcome by matching their internal electrical impedance to that of the applied resistance load. However, the applied resistance load can vary significantly in time, since it depends on the vibration frequency and the working temperature. Hence, a real-time tracking of the applied impedance load should be done to always harvest the maximum energy from the PEH. This paper faces the above problem by presenting an active control able to track and follow in time the optimal working point of a PEH. It exploits a non-conventional AC-DC converter, which integrates a single-stage DC-DC Zeta converter and a full-bridge active rectifier, controlled by a dedicated algorithm based on pulse-width modulation (PWM) with maximum power point tracking (MPPT). A prototype of the proposed converter, based on discrete components, was created and experimentally tested by applying a sudden variation of the resistance load, aimed to emulate a change in the excitation frequency from 30 to 70 Hz and a change in the operating temperature from 25 to 50 °C. Results showed the effectiveness of the proposed approach, which allowed to match the optimal load after 0.38 s for a ΔR of 47 kΩ and after 0.15 s for a ΔR of 18 kΩ.
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http://dx.doi.org/10.3390/s20205862DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589673PMC
October 2020

Cysteine Oxidations in Mitochondrial Membrane Proteins: The Case of VDAC Isoforms in Mammals.

Front Cell Dev Biol 2020 4;8:397. Epub 2020 Jun 4.

Organic Mass Spectrometry Laboratory, Department of Chemical Sciences, University of Catania, Catania, Italy.

Cysteine residues are reactive amino acids that can undergo several modifications driven by redox reagents. Mitochondria are the source of an abundant production of radical species, and it is surprising that such a large availability of highly reactive chemicals is compatible with viable and active organelles, needed for the cell functions. In this work, we review the results highlighting the modifications of cysteines in the most abundant proteins of the outer mitochondrial membrane (OMM), that is, the voltage-dependent anion selective channel (VDAC) isoforms. This interesting protein family carries several cysteines exposed to the oxidative intermembrane space (IMS). Through mass spectrometry (MS) analysis, cysteine posttranslational modifications (PTMs) were precisely determined, and it was discovered that such cysteines can be subject to several oxidization degrees, ranging from the disulfide bridge to the most oxidized, the sulfonic acid, one. The large spectra of VDAC cysteine oxidations, which is unique for OMM proteins, indicate that they have both a regulative function and a buffering capacity able to counteract excess of mitochondrial reactive oxygen species (ROS) load. The consequence of these peculiar cysteine PTMs is discussed.
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http://dx.doi.org/10.3389/fcell.2020.00397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7287182PMC
June 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.
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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.
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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.
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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.
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http://dx.doi.org/10.1016/j.jprot.2019.103530DOI Listing
January 2020

Enhancing grain size in durum wheat using RNAi to knockdown GW2 genes.

Theor Appl Genet 2019 Feb 13;132(2):419-429. Epub 2018 Nov 13.

Department of Agriculture and Forest Sciences, University of Tuscia, Via S. Camillo de Lellis, 01100, Viterbo, Italy.

Key Message: Knocking down GW2 enhances grain size by regulating genes encoding the synthesis of cytokinin, gibberellin, starch and cell wall. Raising crop yield is a priority task in the light of the continuing growth of the world's population and the inexorable loss of arable land to urbanization. Here, the RNAi approach was taken to reduce the abundance of Grain Weight 2 (GW2) transcript in the durum wheat cultivar Svevo. The effect of the knockdown was to increase the grains' starch content by 10-40%, their width by 4-13% and their surface area by 3-5%. Transcriptomic profiling, based on a quantitative real-time PCR platform, revealed that the transcript abundance of genes encoding both cytokinin dehydrogenase 1 and the large subunit of ADP-glucose pyrophosphorylase was markedly increased in the transgenic lines, whereas that of the genes encoding cytokinin dehydrogenase 2 and gibberellin 3-oxidase was reduced. A proteomic analysis of the non-storage fraction extracted from mature grains detected that eleven proteins were differentially represented in the transgenic compared to wild-type grain: some of these were involved, or at least potentially involved, in cell wall development, suggesting a role of GW2 in the regulation of cell division in the wheat grain.
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http://dx.doi.org/10.1007/s00122-018-3229-9DOI Listing
February 2019

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

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

Comparative proteomic analysis of two transgenic low-gliadin wheat lines and non-transgenic wheat control.

J Proteomics 2017 08 15;165:102-112. Epub 2017 Jun 15.

Department of Plant Breeding, Institute for Sustainable Agriculture (IAS-CSIC), 14004 Córdoba, Spain. Electronic address:

Gluten proteins are major determinants of the bread making quality of wheat, but also of important wheat-related disorders, including coeliac disease (CD), and allergies. We carried out a proteomic study using the total grain proteins from two low-gliadin wheat lines, obtained by RNAi, and the untransformed wild type as reference. The impact of silencing on both target and on non-target proteins was evaluated. Because of the great protein complexity, we performed separate analyses of four kernel protein fractions: gliadins and glutenin subunits, and metabolic and CM-like proteins, by using a classical 2D electrophoresis gel based approach followed by RP-HPLC/nESI-MS/MS. As a result of the strong down-regulation of gliadins, the HMW-GS, metabolic and chloroform/methanol soluble proteins were over-accumulated in the transgenic lines, especially in the line D793, which showed the highest silencing of gliadins. Basing on these data, and considering that metabolic proteins and chloroform/methanol soluble proteins (CM-like), such as the α-amylase/trypsin inhibitor family, β-amylase and serpins, were related to wheat allergens, further in vivo analysis will be needed, especially those related to clinical trials in controlled patients, to determine if these lines could be used for food preparation for celiac or other gluten intolerant groups.

Biological Significance: Several enteropathies and allergies are related to wheat proteins. Biotechnological techniques such as genetic transformation and RNA interference have allowed the silencing of gliadin genes, providing lines with very low gliadin content in the grains. We report a proteomic-based approach to characterize two low-gliadin transgenic wheat lines obtained by RNAi technology. These lines harbor the same silencing fragment, but driven by two different endosperm specific promoters (γ-gliadin and D-hordein). The comprehensive proteome analysis of these transgenic lines, by combining two-dimensional electrophoresis and RP-HPLC/nESI-MS/MS, provided a large number of spots differentially expressed between the control and the transgenic lines. Hence, the results of this study will facilitate further safety evaluation of these transgenic lines.
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http://dx.doi.org/10.1016/j.jprot.2017.06.010DOI Listing
August 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.
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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 .
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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.
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http://dx.doi.org/10.1007/s00726-016-2315-zDOI Listing
December 2016

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

VDAC3 as a sensor of oxidative state of the intermembrane space of mitochondria: the putative role of cysteine residue modifications.

Oncotarget 2016 Jan;7(3):2249-68

Department of Biomedicine and Biotechnology BIOMETEC, Section of Biology and Genetics, University of Catania, Catania, Italy.

Voltage-Dependent Anion selective Channels (VDAC) are pore-forming mitochondrial outer membrane proteins. In mammals VDAC3, the least characterized isoform, presents a set of cysteines predicted to be exposed toward the intermembrane space. We find that cysteines in VDAC3 can stay in different oxidation states. This was preliminary observed when, in our experimental conditions, completely lacking any reducing agent, VDAC3 presented a pattern of slightly different electrophoretic mobilities. This observation holds true both for rat liver mitochondrial VDAC3 and for recombinant and refolded human VDAC3. Mass spectroscopy revealed that cysteines 2 and 8 can form a disulfide bridge in native VDAC3. Single or combined site-directed mutagenesis of cysteines 2, 8 and 122 showed that the protein mobility in SDS-PAGE is influenced by the presence of cysteine and by the redox status. In addition, cysteines 2, 8 and 122 are involved in the stability control of the pore as shown by electrophysiology, complementation assays and chemico-physical characterization. Furthermore, a positive correlation between the pore conductance of the mutants and their ability to complement the growth of porin-less yeast mutant cells was found. Our work provides evidence for a complex oxidation pattern of a mitochondrial protein not directly involved in electron transport. The most likely biological meaning of this behavior is to buffer the ROS load and keep track of the redox level in the inter-membrane space, eventually signaling it through conformational changes.
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http://dx.doi.org/10.18632/oncotarget.6850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4823033PMC
January 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.
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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.
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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.
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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.
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http://dx.doi.org/10.1255/ejms.1230DOI Listing
April 2014

The mitochondrial Italian Human Proteome Project initiative (mt-HPP).

Mol Biosyst 2013 Aug 28;9(8):1984-92. Epub 2013 May 28.

Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome, Italy.

Mitochondria carry maternally inherited genetic material, called the mitochondrial genome (mtDNA), which can be defined as the 25th human chromosome. The chromosome-centric Human Proteome Project (c-HPP) has initially focused its activities addressing the characterization and quantification of the nuclear encoded proteins. Following the last International HUPO Congress in Boston (September 2012) it was clear that however small the mitochondrial chromosome is, it plays an important role in many biological and physiopathological functions. Mutations in the mtDNA have been shown to be associated with dozens of unexplained disorders and the information contained in the mtDNA should be of major relevance to the understanding of many human diseases. Within this paper we describe the Italian initiative of the Human Proteome Project dedicated to mitochondria as part of both programs: chromosome-centric (c-HPP) and Biology/Disease (B/D-HPP). The mt-HPP has finally shifted the attention of the HUPO community outside the nuclear chromosomes with the general purpose to highlight the mitochondrial processes influencing the human health. Following this vision and considering the large interest and evidence collected on the non-Mendelian heredity of Homo sapiens associated with mt-chromosome and with the microbial commensal ecosystem constituting our organism we may speculate that this program will represent an initial step toward other HPP initiatives focusing on human phenotypic heredity.
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http://dx.doi.org/10.1039/c3mb70065hDOI Listing
August 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.
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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.
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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.
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http://dx.doi.org/10.1002/mas.20347DOI Listing
October 2012

Development and validation of a liquid chromatography/electrospray ionization tandem mass spectrometry method for the quantification of latanoprost free acid in rabbit aqueous humor and ciliary body.

J Mass Spectrom 2011 Nov;46(11):1168-74

SIFI SpA, Via Ercole Patti 36, 95020 Lavinaio - Aci S. Antonio, Catania, Italy.

A rapid, selective and sensitive method for quantification of latanoprost free acid in rabbit aqueous humor (AH) and ciliary body (CB) using reverse phase-high performance liquid chromatography coupled with electrospray ionization (ESI)-mass spectrometry/mass spectrometry has been developed and validated. Quantification in AH and CB was achieved by stable isotope dilution employing tetra-deuterated analog of latanoprost free acid, used as internal standard. Sample preparation was based on protein precipitation with methanol in AH, and on liquid extraction with a mixture of ethyl acetate and isopropanol 60:40 (v/v) in CB. Elution was achieved on an octylsilica (C8) column, using an isocratic elution method. Detection was performed on a triple quadrupole mass spectrometer, using ESI in positive ion selected reaction monitoring mode. Calibration curves were linear in the validated concentration ranges of 10-160 ng/mL in AH and 80-1280 ng/g in CB. The accuracy and precision values, obtained from three different sets of quality control samples, each analyzed in triplicate on three different days, were within the generally accepted criteria for analytical methods (< 15%). The limit of detection was 30.66 pg/mL in AH and 237.75 pg/g in CB. The assay proved to be accurate and precise when applied to the in vivo study of latanoprost free acid in rabbit AH and CB after single administration of an eye drops containing latanoprost.
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http://dx.doi.org/10.1002/jms.2004DOI Listing
November 2011

Review: applications of mass spectrometry techniques in the investigation of milk proteome.

Eur J Mass Spectrom (Chichester) 2011 ;17(4):305-20

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

The introduction of "soft" desorption/ionization methods such as electrospray ionization and matrix-assisted laser desorption/ionization has determined a breakthrough in the application of mass spectrometry to the structural analysis of proteins. The contemporary advancement of bioinformatics, together with the possibility to combine these mass spectrometric methods with electrophoretic or chromatographic separation techniques has opened up the new field of proteome analysis and, more generally, has established these approaches as indispensable tools for protein and peptide analysis in complex mixtures, such as milk and milk- derived foods. Here, a necessarily not exhaustive series of current applications of mass spectrometry-based techniques for the characterization of milk proteins will be summarized. These include the characterization of milk protein polymorphism, determination of the structural modifications induced on milk proteins by industrial processes, investigation of milk adulterations and characterization of milk allergens.
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http://dx.doi.org/10.1255/ejms.1147DOI Listing
January 2012