Publications by authors named "Antonio Malorni"

27 Publications

  • Page 1 of 1

Use of solid-phase microextraction coupled to gas chromatography-mass spectrometry for determination of urinary volatile organic compounds in autistic children compared with healthy controls.

Anal Bioanal Chem 2014 Jul 15;406(19):4649-62. Epub 2014 May 15.

Institute of Food Science, National Council of Research, via Roma 64, 83100, Avellino, Italy,

Autism spectrum disorders (ASDs) are a group of neurodevelopmental disorders which have a severe life-long effect on behavior and social functioning, and which are associated with metabolic abnormalities. Their diagnosis is on the basis of behavioral and developmental signs usually detected before three years of age, and there is no reliable biological marker. The objective of this study was to establish the volatile urinary metabolomic profiles of 24 autistic children and 21 healthy children (control group) to investigate volatile organic compounds (VOCs) as potential biomarkers for ASDs. Solid-phase microextraction (SPME) using DVB/CAR/PDMS sorbent coupled with gas chromatography-mass spectrometry was used to obtain the metabolomic information patterns. Urine samples were analyzed under both acid and alkaline pH, to profile a range of urinary components with different physicochemical properties. Multivariate statistics techniques were applied to bioanalytical data to visualize clusters of cases and to detect the VOCs able to differentiate autistic patients from healthy children. In particular, orthogonal projections to latent structures discriminant analysis (OPLS-DA) achieved very good separation between autistic and control groups under both acidic and alkaline pH, identifying discriminating metabolites. Among these, 3-methyl-cyclopentanone, 3-methyl-butanal, 2-methyl-butanal, and hexane under acid conditions, and 2-methyl-pyrazine, 2,3-dimethyl-pyrazine, and isoxazolo under alkaline pH had statistically higher levels in urine samples from autistic children than from the control group. Further investigation with a higher number of patients should be performed to outline the metabolic origins of these variables, define a possible association with ASDs, and verify the usefulness of these variables for early-stage diagnosis.
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http://dx.doi.org/10.1007/s00216-014-7855-zDOI Listing
July 2014

Proteomic analysis of peritoneal fluid of patients treated by peritoneal dialysis: effect of glucose concentration.

Nephrol Dial Transplant 2011 Jun 15;26(6):1990-9. Epub 2010 Nov 15.

Department of Internal Medicine, Second University of Naples, Naples, Italy.

Background: Depending on both membrane composition and solute transport rate across the membrane, protein composition of the dialysate of patients receiving peritoneal dialysis (PD) has recently become of great interest. Unfortunately, thus far few studies have focused on dialysate characterization, and further investigations are required to better understand the biological mechanisms influencing PD efficiency.

Methods: Different classical proteomic approaches were combined with advanced mass spectrometric (MS) techniques to analyse peritoneal fluid (PF) protein composition of adult patients receiving PD. Characterization was performed by using 1D gel electrophoresis combined with nano-RP-HPLC-ESI-MS/MS and shotgun proteomics, while comparative analyses were performed coupling 2D gel electrophoresis with MALDI-TOF MS.

Results: The study allowed the identification of 151 different proteins from PF, which are mainly of plasmatic origin. Comparison of PD effluents characterized by different glucose concentrations demonstrated four proteins (apolipoprotein A-IV, fibrinogen beta chain, transthyretin and alpha-1-antitrypsin) to be under-expressed in the highest osmolar solution having 4.25% compared to others having 1.5% and 2.5% glucose. All of them were found to be involved in the inflammatory processes.

Conclusions: This study provides a possible platform for future diagnostic and therapeutic applications in the field of PD and allowed the identification of potential targets to be used in preventing inflammatory processes induced by the exposure to dialysis solutions.
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http://dx.doi.org/10.1093/ndt/gfq670DOI Listing
June 2011

Proteomics for the elucidation of cold adaptation mechanisms in Listeria monocytogenes.

J Proteomics 2010 Sep 8;73(10):2021-30. Epub 2010 Jul 8.

Proteomic and Biomolecular Mass Spectrometry Center, Institute of Food Science, CNR, Avellino, Italy.

Listeria monocytogenes, one of the major food-related pathogens, is the aetiological agent of listeriosis, a potentially life-threatening illness. It is able to survive in hostile environments and stress conditions such as those encountered in food-processing technologies (high salt concentration, wide range of pH and temperature, low water availability) and it also thrives at temperatures ranging from -0.4 to 45 °C. In this study, expression proteomics was applied to gain insight into key cellular events that allow L. monocytogenes to survive and multiply even at refrigeration temperatures. Interestingly, we observed that the adaptation processes mainly affect biochemical pathways related to protein synthesis and folding, nutrient uptake and oxidative stress. Furthermore, proteins implicated in metabolic pathways for energy production, such as glycolysis and Pta-AckA pathway, were present to a higher level in the cells grown at 4 °C. This suggests that, on the whole, cells exhibit an enhanced demand for energy to sustain cold growth. Proteomics may represent a key tool in deciphering specific mechanisms underlying cold adaptation response and, more widely, cell machinery.
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http://dx.doi.org/10.1016/j.jprot.2010.06.011DOI Listing
September 2010

Fish authentication by MALDI-TOF mass spectrometry.

J Agric Food Chem 2008 Dec;56(23):11071-6

Centro di Spettrometria di Massa Proteomica e Biomolecolare, Istituto di Scienze dell'Alimentazione del CNR, Avellino, Italy.

Recent EU directives and regulations for quality control and authentication of food products have prompted the development of new methods for large-scale tests to ensure the protection of consumers. In view of this, an innovative method based on MALDI-TOF mass spectrometry has been developed and successfully applied to fish authentication. Highly specific mass spectrometric profiles from 25 different fish species were obtained. Signals generated from proteins with molecular weights of about 11 kDa have been selected as specific biomarkers for unambiguous discrimination. This method is also suitable for verifying commercial product authenticity and to rapidly discriminate species subjected to fraudulent substitutions, such as those belonging to Gadidae and Pleuronectiformes. For example, biomarkers for fillets of sole (m/z 11975.21), European plaice (m/z 11351.73, 11763.63) and Greenland halibut (m/z 11432.38) were defined. Structural characterization by mass spectrometry of several proteins generating biomarker signals allowed us to identify them as parvalbumins, known to be among the major fish allergens.
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http://dx.doi.org/10.1021/jf8021783DOI Listing
December 2008

Mass spectrometric and linear discriminant analysis of N-glycans of human serum alpha-1-acid glycoprotein in cancer patients and healthy individuals.

J Proteomics 2008 Jul 2;71(2):186-97. Epub 2008 May 2.

Institute of Structural Chemistry, Chemical Research Center, Hungarian Academy of Sciences, Budapest, Pusztaszeri u.59-67, Hungary.

N-glycan oligosaccharides of human serum alpha(1)-acid glycoprotein (AGP) samples isolated from 43 individuals (healthy individuals and patients with lymphoma and with ovarian tumor) were analyzed by MALDI-TOF mass spectrometry and a multivariate statistical method (linear discriminant analysis, LDA). 34 different glycan structures have been identified. From the glycosylation pattern determined by mass spectrometry fucosylation and branching indices have been calculated. These parameters show only small differences between the patient groups studied, but these differences are not sufficiently large to use as a potential biomarker. LDA analysis, on the other hand shows a very good separation between the three groups (with a classification of 88%). Cross-validation indicates that the method has predictive power: Identifying cancerous vs. healthy individuals shows 96% selectivity and 93% specificity; identification of lymphoma vs. the mixed group of healthy and ovarian tumor cases is also promising (72% selectivity and 84% specificity). The pilot study presented here demonstrates that mass spectrometry combined with linear discriminant analysis (LDA) may provide valuable data for identifying and studying the pathophysiology of malignant diseases.
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http://dx.doi.org/10.1016/j.jprot.2008.04.005DOI Listing
July 2008

Proteomic analysis of exoproteins expressed by enterotoxigenic Staphylococcus aureus strains.

Proteomics 2008 Jun;8(12):2462-76

Proteomic and Biomolecular Mass Spectrometry Center (CeSMa-ProBio), Institute of Food Science and Technology, C.N.R., Avellino, Italy.

Pathogenic bacteria excrete a variety of virulence factors into extracellular medium and to the cell surface which have essential roles in the colonization and insurrection of the host cells, and thus reflect the degree of bacterial pathogenicity. For the exploration of virulence factors expressed in the secreted proteome fraction, different Staphylococcus aureus strains were analyzed using gel-based bottom-up proteomic approach. A total of 119 distinct proteins were identified for the enterotoxin gene cluster (egc) negative and seb gene positive S. aureus American Type Culture Collection (ATCC) 14458 strain by the use of one- and 2-DE based proteomics. Detailed analysis of enterotoxin region of the 2-D map confirmed, beside the highly expressed staphylococcal enterotoxin B (SEB), the presence of enterotoxin-like proteins SElK and SElQ previously predicted by genotyping (Sergeev et al.., J. Clin. Microbiol. 2004, 42, 2134-2143). Exoprotein patterns at the late-exponential (7 h) and stationary (24 h) phases of cellular growth show a high-level similarity in this region. Comparative analysis of enterotoxin region of five S. aureus strains including two clinical isolates (RIMD 31092 and A900322), a food derived strain (AB-8802) with highly prevalent egc positive operon and a nonenterotoxigenic reference strain (ROS) revealed the presence of different known enterotoxins and other virulence factors along with a number of core exoproteins. In addition, production of SElL (RIMD 31092) and SElP (A900322) was demonstrated for the first time at the protein level. Under the experimental conditions applied none of the enterotoxins encoded by the genes of egc operon was identified.
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http://dx.doi.org/10.1002/pmic.200700965DOI Listing
June 2008

DNA oxidation as triggered by H3K9me2 demethylation drives estrogen-induced gene expression.

Science 2008 Jan;319(5860):202-6

Istituto di Scienze dell'Alimentazione, Consiglio Nazionale delle Ricerche (C.N.R.), 83100 Avellino, Italy.

Modifications at the N-terminal tails of nucleosomal histones are required for efficient transcription in vivo. We analyzed how H3 histone methylation and demethylation control expression of estrogen-responsive genes and show that a DNA-bound estrogen receptor directs transcription by participating in bending chromatin to contact the RNA polymerase II recruited to the promoter. This process is driven by receptor-targeted demethylation of H3 lysine 9 at both enhancer and promoter sites and is achieved by activation of resident LSD1 demethylase. Localized demethylation produces hydrogen peroxide, which modifies the surrounding DNA and recruits 8-oxoguanine-DNA glycosylase 1 and topoisomeraseIIbeta, triggering chromatin and DNA conformational changes that are essential for estrogen-induced transcription. Our data show a strategy that uses controlled DNA damage and repair to guide productive transcription.
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http://dx.doi.org/10.1126/science.1147674DOI Listing
January 2008

Proteomic investigation of the aggregation phenomenon in Lactobacillus crispatus.

Biochim Biophys Acta 2008 Feb 22;1784(2):335-42. Epub 2007 Nov 22.

Centro di Spettrometria di Massa Proteomica e Biomolecolare, Istituto di Scienze dell'Alimentazione del CNR, Avellino, Italy.

Aggregation process affects the ability of Lactobacillus crispatus, a probiotic, to survive into the gastro-intestinal environment and to adhere to the intestinal mucosa. To elucidate mechanisms underlying this process, a comparative proteomic study was carried out on a wild type strain M247 and its spontaneous isogenic mutant Mu5, which had lost the aggregative phenotype. Results highlighted an overall lower amount of enzymes involved in carbohydrate transport and metabolism in strain M247 compared to strain Mu5, suggesting a reduction in the general growth rate, probably caused by nutrient limitation in cell aggregates, coherently with the phenotypic traits of the strains. Moreover, the up-regulation of a putative elongation factor Tu in the wild type M247 strain could suggest a role of this particular protein in the adhesion mechanism of L. crispatus.
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http://dx.doi.org/10.1016/j.bbapap.2007.11.007DOI Listing
February 2008

Identification of a peptide from alpha-gliadin resistant to digestive enzymes: implications for celiac disease.

J Chromatogr B Analyt Technol Biomed Life Sci 2007 Aug 18;855(2):236-41. Epub 2007 May 18.

Proteomic and Biomolecular Mass Spectrometry Centre, Institute of Food Science CNR, Avellino, Italy.

Current knowledge indicates that both innate and adaptive immune responses are involved in Celiac disease (CD) driven by different gliadin peptides. By studying a representative recombinant alpha-gliadin form, a further 25-mer peptide resistant to gastric, pancreatic, and human intestinal brush-border membrane enzymes was detected. This peptide latter encompasses the sequence 31-43 known to elicit the innate immune response in CD. The resistance of 25-mer, as well as that of the already described 33-mer related to the CD adaptive immune response, was confirmed on a standard flour wheat sample representative of the most widespread European varieties.
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http://dx.doi.org/10.1016/j.jchromb.2007.05.009DOI Listing
August 2007

Survey of polychlorinated dibenzo-p-dioxins (PCDDs), Polychlorinated Dibenzo-p-furans (PCDFs), polychlorinated biphenyls (PCBs), and mineral components in Italian citrus cold-pressed essential oils.

J Agric Food Chem 2007 Feb 24;55(4):1627-37. Epub 2007 Jan 24.

Laboratorio Diossine e Microinquinanti Organici, Stazione Sperimentale per le Industrie delle Essenze e dei derivati dagli Agrumi (SSEA), via Gen. Tommasini 2, 89127 Reggio Calabria, Italy.

Investigation of PCDDs, PCDFs, PCBs, Al, As, Pb, Ba, Co, Cu, Cr, Fe, Mn, Cd, Ag, Sn, Zn, and Hg contents in 60 samples of cold-pressed essential oils produced in Calabria and Sicily in 2003-2005 was carried out. PCDDs, PCDFs, and PCBs were analyzed by HRGC-HRMS techniques using U.S. EPA 1613/94 and U.S. EPA 1668/A (1999) analytical methods. Mineral components were determined through GFAAS techniques; Hg content was determined by FI-M/H-AAS. The results of this study showed that essential oil contamination was due to a widespread pollution, typical background of rural areas, with relatively higher concentrations of PCDDs compared to PCDFs and little presence of PeCDF. Congeners OCDD, HpCDF, and OCDF were found at high concentrations. Regarding mineral components, mean values of Cr, Fe, and Ni were in agreement with data reported in the literature. Concentrations of As and Pb were below the maximum limits accepted by the current legislation. Finally, none of the samples analyzed were contaminated with Hg.
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http://dx.doi.org/10.1021/jf062622vDOI Listing
February 2007

Influence of winemaking practices on the concentration of rare earth elements in white wines studied by inductively coupled plasma mass spectrometry.

J Agric Food Chem 2007 Jan;55(2):311-7

Proteomic and Biomolecular Mass Spectrometry Center (CeSMa-ProBio), Institute of Food Science and Technology, C.N.R., via Roma, 52 a/c, 83100 Avellino, Italy.

Influence of clarification, filtration, and storage on the concentration of rare earth elements (REEs) was studied in white wines by inductively coupled plasma mass spectrometry (ICP-MS). Smooth and parallel chondrite-normalized (CN) plots were obtained for wines which have never been in contact with fining agents. Clarification and filtration generally used in white wine production were simulated in the laboratory using nontreated reference wines, and CN plots were compared before and after treatments. Clarification by bentonites yields an overall increase in REE concentrations resulting in substantially parallel CN curves well above the plots of the corresponding nontreated wines. Filtration using silicate (SiO2), on the other hand, changes the CN profile in a nonparallel manner due to a higher release of La, Ce, Pr, Nd, and Gd, more than other elements studied. Filtration with cellulose powder causes a small increase in the concentration of light REEs, while the concentrations of other elements remain basically unchanged. Storage conditions could also affect the REE pattern of wine. We found that the influence of glass is greater than that of stainless steel and wood. In addition, we report that commercially available finished white wines from the same region show highly different REE patterns depending on the winemaking practices employed.
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http://dx.doi.org/10.1021/jf061828tDOI Listing
January 2007

Phosphorylation of B14.5a subunit from bovine heart complex I identified by titanium dioxide selective enrichment and shotgun proteomics.

Mol Cell Proteomics 2007 Feb 17;6(2):231-7. Epub 2006 Nov 17.

Proteomic and Biomolecular Mass Spectrometry Centre, Institute of Food Science and Technology, Consiglio Nazionale delle Ricerche, 83100 Avellino, Italy.

Shotgun proteomics was used to study the steady phosphorylation state of NADH:ubiquinone oxidoreductase (complex I) subunits from bovine heart mitochondria. A total tryptic digestion of enzymatically active complex I was performed, and the resulting peptide mixture was subjected to phosphopeptide enrichment by the use of titanium dioxide (TiO2). The phosphopeptide-enriched fraction was separated and analyzed with nanoscale reverse-phase HPLC-ESI-MS/MS in single information-dependent acquisition. Hence two phosphorylated complex I subunits were detected: 42 kDa and B14.5a. Phosphorylation of 42-kDa subunit at Ser-59 has already been determined with fluorescent phosphoprotein-specific gel staining and mass spectrometry (Schilling, B., Aggeler, R., Schulenberg, B., Murray, J., Row, R. H., Capaldi, R. A., and Gibson, B. W. (2005) Mass spectrometric identification of novel phosphorylation site in subunit NDUFA10 of bovine mitochondrial complex I. FEBS Lett. 579, 2485-2490). In our work, this finding was confirmed using a non-gel-based approach. In addition, we report novel phosphorylation on B14.5a nuclear encoded subunit. We demonstrated evidence of the phosphorylation site at Ser-95 residue by collision-induced dissociation experiments on three different molecular ions of two tryptic phosphopeptides of B14.5a.
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http://dx.doi.org/10.1074/mcp.M600268-MCP200DOI Listing
February 2007

Proteomic analysis of MCF-7 breast cancer cell line exposed to mitogenic concentration of 17beta-estradiol.

Proteomics 2006 Nov;6(22):5973-82

Proteomic and Biomolecular Mass Spectrometry Center (CeSMa-ProBio), Institute of Food Science and Technology, C.N.R., Avellino, Italy.

Estrogens are powerful mitogens that play a critical role in the onset of breast cancer and its progression. About two-thirds of all breast cancers are estrogen receptor (ER)+ at the time of diagnosis, and the ER expression is the determinant of a tumor phenotype associated with hormone responsiveness. The molecular basis of the relationship between ER expression, (anti)hormonal responsiveness, and breast cancer prognosis is still unknown. To identify the proteins affected by the presence of the hormone we used 2-D-PAGE-based bottom-up proteomics for the study of the proteome of MCF-7 cells of estrogen-responsive breast carcinoma exposed to a mitogenic concentration of 17beta-estradiol (E2) for 12, 18, 24, and 30 h. Differential expression analysis showed significant changes for 12 proteins. These include ezrin-radixin-moesin-binding phosphoprotein of 50 kDa which was previously shown to be directly regulated by E2. Expression profiles of other proteins already implicated in the progression of breast cancer, such as stathmin, calreticulin, heat shock 71 kDa, alpha-enolase are also described. Moreover, it is observed that different unexpected proteins, translation factors, and energetic metabolism enzymes are also influenced by the presence of the hormone.
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http://dx.doi.org/10.1002/pmic.200600333DOI Listing
November 2006

Shotgun proteomics for the characterization of subunit composition of mitochondrial complex I.

Biochim Biophys Acta 2006 Sep-Oct;1757(9-10):1438-50. Epub 2006 Jun 3.

Proteomic and Biomolecular Mass Spectrometry Centre, Institute of Food Science and Technology, CNR, via Roma 52, a/c, 83100 Avellino, Italy.

Here we propose shotgun proteomics as an alternative method to gel-based bottom-up proteomic platform for the structural characterization of mitochondrial NADH:ubiquinone oxidoreductase (complex I). The approach is based on simultaneous identification of subunits after global digestion of the intact complex. Resulting mixture of tryptic peptides is purified, concentrated, separated and online analyzed using nano-scale reverse-phase nano-ESI-MS/MS in a single information dependent acquisition mode. The usefulness of the method is demonstrated in our work on the well described model system of complex I from bovine heart mitochondria. The shotgun method led to the identification and partial sequence characterization of 42 subunits representing more than 95% coverage of the complex. In particular, almost all nuclear (except MLRQ) and 5 mitochondria DNA encoded subunits (except ND4L and ND6) were identified. Furthermore, it was possible to identify 30 co-purified proteins of the inner mitochondrial membrane structurally not belonging to complex I. The method's efficiency is shown by comparing it to two classical 1 D gel-based strategies. Shotgun proteomics is less laborious, significantly faster and requires less sample material with minimal treatment, facilitating the screening for post-translational modifications and quantitative and qualitative differences of complex I subunits in genetic disorders.
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http://dx.doi.org/10.1016/j.bbabio.2006.05.037DOI Listing
December 2006

Proteomic analysis of MCF-7 cell lines expressing the zinc-finger or the proline-rich domain of retinoblastoma-interacting-zinc-finger protein.

J Proteome Res 2006 May;5(5):1176-85

Dipartimento di Scienze della Vita, Seconda Università degli Studi di Napoli, I-81100 Caserta, Italy.

To identify a growth-promoting activity related to retinoblastoma-interacting-zinc-finger (RIZ) protein, differential protein expression of MCF-7 cell lines expressing the zinc-finger or the proline-rich domain of RIZ protein was analyzed by a robust bottom-up mass-spectrometry proteomic approach. Spots corresponding to qualitative and quantitative differences in protein expression have been selected and identified. Some of these proteins have been previously reported as being associated with different types of carcinomas or involved in cell proliferation and differentiation. Knowledge of specific differentially expressed proteins by MCF-7-derived cell lines expressing RIZ different domains will provide the basis for identifying a growth-promoting activity related to RIZ gene products.
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http://dx.doi.org/10.1021/pr0504743DOI Listing
May 2006

Matrix-assisted laser desorption ionization-time of flight mass spectrometry for the discrimination of food-borne microorganisms.

Appl Environ Microbiol 2006 Feb;72(2):1180-9

Proteomic and Biomolecular Mass Spectrometry Center, Institute of Food Science and Technology, CNR, via Roma 52 a/c, 83100 Avellino, Italy.

A methodology based on matrix-assisted laser desorption ionization-time of flight mass spectrometry of intact bacterial cells was used for rapid discrimination of 24 bacterial species, and detailed analyses to identify Escherichia coli O157:H7 were carried out. Highly specific mass spectrometric profiles of pathogenic and nonpathogenic bacteria that are well-known major food contaminants were obtained, uploaded in a specific database, and made available on the Web. In order to standardize the analytical protocol, several experimental, sample preparation, and mass spectrometry parameters that can affect the reproducibility and accuracy of data were evaluated. Our results confirm the conclusion that this strategy is a powerful tool for rapid and accurate identification of bacterial species and that mass spectrometric methodologies could play an essential role in polyphasic approaches to the identification of pathogenic bacteria.
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http://dx.doi.org/10.1128/AEM.72.2.1180-1189.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1392959PMC
February 2006

MALDI-TOF mass spectrometry of a combinatorial peptide library: effect of matrix composition on signal suppression.

J Mass Spectrom 2005 Dec;40(12):1590-4

Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eötvös L. University, P. O. Box 32, Budapest 112, Hungary, H-1518.

The effect of matrix composition on signal suppression caused by a dominant compound under MALDI ionization was studied using the combinatorial TQTXT pentapeptide library as a model system. The peptide library is composed of 19 components with all proteinogenic amino acids except cysteine in position X. From these compounds, only the Arg peptide (TQTRT) was detected with sufficient intensity in the MALDI-TOF mass spectrum under typical MALDI conditions (CCA matrix). The analysis of a set of compounds utilized as different matrix components, additives and a cationizing agent revealed that the composition of the matrix is a critical point in signal suppression. Highly improved ion yields were achieved by using a CCA/DHB mixture as a matrix. The addition of K(+) as a cationizing agent to the CCA matrix resulted in MALDI-TOF mass spectra with relative ion intensities very similar to those obtained by electrospray ionization.
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http://dx.doi.org/10.1002/jms.937DOI Listing
December 2005

Glycosylation site analysis of human alpha-1-acid glycoprotein (AGP) by capillary liquid chromatography-electrospray mass spectrometry.

J Mass Spectrom 2005 Nov;40(11):1472-83

Department of Mass Spectrometry, Institute of Structural Chemistry, Chemical Research Center, Hungarian Academy of Sciences, 1525 Budapest, Hungary.

A new anionic surfactant (RapiGest SF) was successfully used for site-specific analysis of glycosylation in human alpha-1-acid glycoprotein (AGP). By means of this analytical approach combined with capillary HPLC-mass spectrometry (and tandem mass spectrometry), the N-linked glycosylation pattern of AGP was explored. On the basis of mass matching and MS/MS experiments ca 80 different AGP-derived glycopeptides were identified. Glycosylation shows a markedly different pattern for the various glycosylation sites. At sites I and II, triantennary complex-type oligosaccharides predominate and at sites III, IV and V, tetra-antennary complex-type oligosaccharides predominate. Sites IV and V show the presence of additional N-acetyl lactosamine (Gal-GlcNAc) units (even higher degree of branching and/or longer antennae are also present).
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http://dx.doi.org/10.1002/jms.938DOI Listing
November 2005

Combination of solid-phase affinity capture on magnetic beads and mass spectrometry to study non-covalent interactions: example of minor groove binding drugs.

Rapid Commun Mass Spectrom 2005 ;19(22):3307-14

Proteomic and Biomolecular Mass Spectrometry Center (CeSMa-ProBio), Institute of Food Science and Technology, C.N.R., via Roma 52 a/c, 83100 Avellino, Italy.

A simple and novel approach was developed to detect non-covalent interactions. It is based on combination of solid-phase affinity capture with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). One of the interacting molecules is bound to magnetic beads and is incubated with the target molecules in solution. The complex bound on the solid support is removed from the solution and transferred for MALDI analysis. Mass spectrometry is used only to detect the target compound, which is far more straightforward than detecting the intact non-covalent complex. To demonstrate the applicability of the method, an AT-rich oligonucleotide (5'-CCCCCAATTCCCCC-3') and its complementary biotinylated sequence (5'-biotin-GGGGGAATTGGGGG-3') were hybridized and immobilized to paramagnetic particles by streptavidin-biotin interaction. The immobilized duplex oligonucleotide was reacted with minor groove binding drugs, Netropsin, Distamycin A, Hoechst 33258 and 4',6-diamidino-2-phenylindole. The resulting DNA-drug complex bound to the particles was separated and analyzed by linear MALDI-TOFMS after washing. Drugs were selectively detected in the spectra. Relative binding strengths were also estimated using competitive complexation.
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http://dx.doi.org/10.1002/rcm.2193DOI Listing
March 2006

Dietary effects of copper and iron deficiency on rat intestine: a differential display proteome analysis.

J Proteome Res 2005 Sep-Oct;4(5):1781-8

Dipartimento di Scienze Farmaceutiche Università degli Studi di Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA), Italy.

Copper and iron are cofactors of many metallo-proteins that accomplish vital functions, such as oxygen and electron transport. Specific metabolic pathways have been selected through evolution, although still not fully elucidated, to confine the dangerous reactivity of their free ionic forms. Inadequate supply of both metals can severely affect basic physiological functions. A differential analysis of the rat intestinal proteome evidenced the following dietary copper- and iron-deficiencies, i.e., significant changes in the levels of proteins belonging to different functional classes (glucose and fatty acid metabolism, molecular chaperones, cytoskeleton plasticity, vitamin transporters). The presented results bring new perspectives to understand the role of copper and iron in the metabolic pathways and provide novel diagnostic tools to characterize the effects of subclinical deficiencies of both metals in unbalanced nutritional disorders.
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http://dx.doi.org/10.1021/pr0501012DOI Listing
January 2006

Partial characterization of glycosphingolipids of Agelas sponges in their peracetylated form by liquid secondary ionization mass spectrometry and high-performance liquid chromatography combined with direct electrospray ionization mass spectrometric detection.

Rapid Commun Mass Spectrom 2004 ;18(24):2989-96

Department of Chemistry of Natural Products, University of Naples, Via Domenico Montesano, 49, I-80131 Naples, Italy.

Electrospray ionization (ESI) and liquid secondary ionization (LSI) mass spectrometry were applied for characterization of glycosphingolipids (GSLs) isolated in their peracetylated form from four Agelas marine sponge species. Since peracetylated glycosphingolipids are not soluble in solvents traditionally used for ESI, lithium chloride was added to the samples in order to obtain lithium cationized molecules. Although the preferred fragmentation seems to be the sequential loss of acetic acid molecules, it was found that tandem mass spectra obtained from peracetylated diglycosyl ceramides might provide direct information about the structure of the long-chain base (formation of W''/Z0 fragments). The utility of ESI and LSI in the analysis of these compounds has also been compared. It was found that the tandem mass spectra obtained by LSI-MS/MS experiments could provide information about the chain-length (carbon atom number) variations within a certain ceramide mass. Thus, from one of our samples, 25 different ceramide compositions have been identified from 8 precursor (Z0) ions. Comparison of the two ionization modes (LSI and ESI) highlights the fact that molecular mass distributions obtained by LSI-MS, especially the presence of unsaturated species, have to be interpreted carefully. For the first time a direct high-performance liquid chromatography (HPLC)/ESI-MS method was used for characterization of complex mixtures of peracetylated GSLs. The results demonstrate that HPLC/ESI-MS is able to analyze mono- and diglycosylated GSLs, and other kinds of glycolipids that are actually present in the sample.
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http://dx.doi.org/10.1002/rcm.1720DOI Listing
March 2005

Detection of immune complexes by matrix-assisted laser desorption/ionization mass spectrometry.

Rapid Commun Mass Spectrom 2003 ;17(24):2741-7

Chemical Research Center, Hungarian Academy of Sciences, Budapest, Hungary.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to detect an immune complex formed between beta-lactoglobulin and polyclonal anti-beta-lactoglobulin antibody in the gas phase. The most important experimental parameters to detect such a specific antibody-antigen complex by MALDI were the use of solutions at near-neutral pH and of sinapinic acid matrix prepared by the dried-droplet method. Under such conditions, predominantly one but also two molecules of antigen protein were complexed by the antibody. Specific formation of the antibody-antigen complex was confirmed by performing competitive reactions. Addition of antibody to a 1:1 mixture of beta-lactoglobulin and one control protein resulted not only in the appearance of the expected antibody-antigen complex, but also in a strong decrease in the free beta-lactoglobulin signal, while the abundance of the control protein was not influenced.
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http://dx.doi.org/10.1002/rcm.1239DOI Listing
April 2004

Identification of transglutaminase-mediated deamidation sites in a recombinant alpha-gliadin by advanced mass-spectrometric methodologies.

Protein Sci 2003 Nov;12(11):2434-42

Centro di Spettrometria di Massa Proteomica e Biomolecolare, Istituto di Scienze dell'Alimentazione del CNR, Avellino, Italy.

Celiac disease is a permanent immune-mediated food intolerance triggered by ingestion of wheat gliadins in genetically susceptible individuals. It has been reported that tissue transglutaminase plays an important role in the onset of celiac disease by converting specific glutamine residues within gliadin fragments into glutamic acid residues. This process increases binding affinity of gliadin peptides to HLA-DQ2/DQ8 molecules, thus enhancing the immune response. The aim of the present study was to achieve a detailed structural characterization of modifications induced by transglutaminase on gliadin peptides. Therefore, structural analyses were carried out on a recombinant alpha-gliadin and on a panel of 26 synthetic peptides, overlapping the complete protein sequence. Modified glutamine residues were identified by means of advanced mass-spectrometric methodologies on the basis of MALDI-TOF-MS and tandem mass spectrometry. Results led to the identification of 19 of 94 glutamine residues present in the recombinant alpha-gliadin, which were converted into glutamic acid residues by a transglutaminase-mediated reaction. This allowed us to achieve a global view of the modifications induced by the enzyme on this protein. Furthermore, results gathered could likely be utilized as relevant information for a better understanding of processes leading to T-cell recognition of gliadin peptides involved in celiac disease.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2366954PMC
http://dx.doi.org/10.1110/ps.03185903DOI Listing
November 2003

Casein phosphoproteome: identification of phosphoproteins by combined mass spectrometry and two-dimensional gel electrophoresis.

Electrophoresis 2003 Aug;24(16):2824-37

Istituto di Scienze dell'Alimentazione del C.N.R., I-83100 Avellino, Italy.

We report a fast and easy-to-use procedure that combines polyacrylamide gel electrophoresis with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF) and nanoelectrospray-tandem mass spectrometry (nES-MS/MS) analysis for the identification of casein components and defined phosphorylated sites. This methodology ensured identification of more than 30 phosphorylated proteins, five beta-, fifteen alpha(s1)-, ten alpha(s2)-, and four kappa-casein (CN) components, including nonallelic, differently phosphorylated, and glycosylated forms. The sugar motif covalently bound to kappa-CN was identified as chains, trisaccharide GalNAc, Gal, NeuGc, and tetrasaccharide 1GalNAc, 1Gal, 2NeuGc. Also identified was a biantennary chain made up of both chains of trisaccharide 1GalNAc, 1Gal, 1NeuGc, and tetrasaccharide 1GalNAc, 1Gal, 2NeuGc moiety on a single kappa-CN component. The phosphate group on site Ser12 of tryptic peptide 8-22 of most phosphorylated alpha(s1)-CN (11 phosphate groups) was localized and the oligosaccharide sequence of the main tryptic glycopeptides of two kappa-CN components was determined by means of MS/MS analysis.
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http://dx.doi.org/10.1002/elps.200305545DOI Listing
August 2003

Mass spectrometric analysis of combinatorial peptide libraries derived from the tandem repeat unit of MUC2 mucin.

J Pept Sci 2003 Jun;9(6):361-74

Chemical Research Center, Hungarian Academy of Sciences, Budapest, Hungary.

Four 19-member synthetic peptide libraries, based on the TX1TX2T epitope motif of the mucin-2 gastrointestinal glycoprotein (MUC2) and ranging in peptide length from dipeptides to 15-mers (XT, TXT, TQTXT and KVTPTPTPTGTQTXT), were synthesized by combinatorial solid phase peptide synthesis using the portioning-mixing combinatorial approach, and analysed by electrospray ionization mass spectrometry at different (1000-10000) resolutions. Most of the components of the individual libraries could be easily identified in a single-stage molecular mass screening experiment. The resolving power of the instrument becomes an important factor above 800-1000 Da molecular mass, when predominantly multiply charged molecular ions are formed. Approaches to the identification of isobars (glutamine/lysine), isomers leucine/isoleucine) and sequence variations by tandem mass spectrometry, and/or by high-performance liquid chromatography-mass spectrometry are outlined.
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http://dx.doi.org/10.1002/psc.462DOI Listing
June 2003

Triterpenoid saponins from the shells of Argania spinosa seeds.

J Agric Food Chem 2002 Jul;50(16):4600-3

Laboratoire de Chimie des Plantes et de Synthèse Organique et Bioorganique, Faculté des Sciences, University of Rabat, B.P. 1041, RP Rabat, Morocco.

Two new oleanene saponins were isolated from the MeOH extract of the shell of Argania spinosa. They possess protobassic acid and 16alpha-protobassic acid as aglycons. The disaccharide moiety linked to C-3 of the aglycon is made up of two glucose units; the pentasaccharide moiety linked to C-28 is made up of arabinose, xylose, and three rhamnose units. Their structures were elucidated by 1D and 2D NMR experiments including (1)H-(1)H (DQF-COSY, 1D TOCSY, and 2D HOHAHA) and (1)H-(13)C (HSQC and HMBC) spectroscopy along with mass spectrometry.
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http://dx.doi.org/10.1021/jf0200117DOI Listing
July 2002

Structural analysis of styrene oxide/haemoglobin adducts by mass spectrometry: identification of suitable biomarkers for human exposure evaluation.

Rapid Commun Mass Spectrom 2002 ;16(9):871-8

Dipartimento di Medicina Sperimentale, Sezione di Medicina del Lavoro, Igiene e Tossicologia Professionale, Seconda Università di Napoli, Piazza Miraglia 2, I-80134 Napoli, Italy.

The structural characterisation of adducts formed by the in vitro reaction of haemoglobin (Hb) with styrene oxide (SO), the most reactive metabolite of the industrial reagent styrene, was obtained by liquid chromatography/electrospray ionisation mass spectrometry (LC/ES-MS) analysis of modified tryptic peptides of human Hb chains. The reactive sites of human Hb towards SO were identified through characterisation of alkylated tryptic peptides by matrix-assisted laser desorption/ionisation with tandem mass spectrometry (MALDI-MS/MS). A procedure was set up based on this characterisation, allowing Hb modification to be assessed by monitoring SO/Hb adducts using HPLC with selected ion recording (SIR) mass spectrometry. By this methodology it was also possible to compare advantages and disadvantages of presently available strategies for the measurement of Hb adducts with SO. The results obtained could most plausibly lead to the optimisation of molecular dosimetry of SO adducts, and the analytical procedure described herein could be applied to the biological monitoring of styrene exposure in the workplace.
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http://dx.doi.org/10.1002/rcm.655DOI Listing
May 2002
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