Publications by authors named "Federica Iavarone"

75 Publications

Oxidative and Proteolytic Inactivation of Alpha-1 Antitrypsin in Bronchopulmonary Dysplasia Pathogenesis: A Top-Down Proteomic Bronchoalveolar Lavage Fluid Analysis.

Front Pediatr 2021 23;9:597415. Epub 2021 Mar 23.

Dipartimento di Scienze della Vita e Sanità Pubblica, Unità Operativa Complessa di Neonatologia, Fondazione Policlinico Universitario A. Gemelli, Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy.

The study investigates the role of the oxidative and proteolytic inactivation of alpha-1 antitrypsin (AAT) in the pathogenesis of bronchopulmonary dysplasia (BPD) in premature infants. Bronchoalveolar lavage fluid (BALF) samples were collected on the 3rd day of life from mechanically ventilated neonates with gestational age ≤ 30 weeks and analyzed without previous treatment (top-down proteomics) by reverse-phase high-performance liquid chromatography-electrospray ionization mass spectrometry. AAT fragments were identified by high-resolution LTQ Orbitrap XL experiments and the relative abundances determined by considering the extracted ion current (XIC) peak area. Forty preterm neonates were studied: 20 (50%) did not develop BPD (no-BPD group), 17 (42.5%) developed mild or moderate new-BPD (mild + moderate BPD group), and 3 (7.5%) developed severe new-BPD (severe BPD group). Eighteen fragments of AAT and a fragment of AAT oxidized at a methionine residue were identified: significantly higher values of AAT fragments 25-57, 375-418, 397-418, 144-171, and 397-418 with oxidized methionine were found in the severe BPD group. The significantly higher levels of several AAT fragments and of the fragment 397-418, oxidized in BALF of preterm infants developing BPD, underlie the central role of an imbalance between proteases and protease inhibitors in exacerbating lung injury and inducing most severe forms of BPD. The study has some limitations, and between them, the small sample size implies the need for further confirmation by larger studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fped.2021.597415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8021761PMC
March 2021

HPLC-ESI-MS top-down analysis of salivary peptides of preterm newborns evidenced high activity of some exopeptidases and convertases during late fetal development.

Talanta 2021 Jan 25;222:121429. Epub 2020 Aug 25.

Laboratorio di Proteomica, Centro Europeo di Ricerca Sul Cervello, IRCCS Fondazione Santa Lucia, Roma, Italy. Electronic address:

To have information on the proteolytic activity of convertases and exo-peptidases on human salivary proteins, this study investigated the relative amounts of the truncated proteoforms in the saliva of preterm newborns and compared them with the relative amounts measured in saliva of at-term newborns, of babies (0-10 years old) and of adults. Results indicated that convertase(s), acting on acidic proline-rich proteins and histatin 3, and carboxypeptidase(s) acting on acidic proline-rich proteins, P-C peptide, histatin 6 and statherin were many folds more active in preterm newborns than in the other groups. Conversely, the aminopeptidase responsible for the removal of the N-terminal Asp residue of statherin was not active in preterm newborns, becoming active only several months after the normal term of delivery. The high activity of convertases determined in preterm newborns suggests that it is required for the molecular events connected to the fetus development, and encourages further studies devoted to the characterization of their specific substrates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.talanta.2020.121429DOI Listing
January 2021

Mass spectrometry characterization of light chain fragmentation sites in cardiac AL amyloidosis: insights into the timing of proteolysis.

J Biol Chem 2020 12 20;295(49):16572-16584. Epub 2020 Sep 20.

Amyloidosis Research and Treatment Center, Fondazione IRCCS Policlinico San Matteo and University of Pavia, Pavia, Italy.

Amyloid fibrils are polymeric structures originating from aggregation of misfolded proteins. , proteolysis may modulate amyloidogenesis and fibril stability. In light chain (AL) amyloidosis, fragmented light chains (LCs) are abundant components of amyloid deposits; however, site and timing of proteolysis are debated. Identification of the N and C termini of LC fragments is instrumental to understanding involved processes and enzymes. We investigated the N and C terminome of the LC proteoforms in fibrils extracted from the hearts of two AL cardiomyopathy patients, using a proteomic approach based on derivatization of N- and C-terminal residues, followed by mapping of fragmentation sites on the structures of native and fibrillar relevant LCs. We provide the first high-specificity map of proteolytic cleavages in natural AL amyloid. Proteolysis occurs both on the LC variable and constant domains, generating a complex fragmentation pattern. The structural analysis indicates extensive remodeling by multiple proteases, largely taking place on poorly folded regions of the fibril surfaces. This study adds novel important knowledge on amyloid LC processing: although our data do not exclude that proteolysis of native LC dimers may destabilize their structure and favor fibril formation, the data show that LC deposition largely precedes the proteolytic events documentable in mature AL fibrils.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA120.013461DOI Listing
December 2020

Top down proteomic analysis of gingival crevicular fluid in deciduous, exfoliating and permanent teeth in children.

J Proteomics 2020 08 3;226:103890. Epub 2020 Jul 3.

Laboratorio di Proteomica e Metabonomica-IRCCS Fondazione Santa Lucia, Roma, Italy.

Gingival Crevicular Fluid (GCF), a plasma-derived exudate present in the gingival crevice was collected from deciduous, exfoliating and permanent teeth from 20 children (60 samples) with the aim to characterize and quantify by a mass spectrometry based top-down proteomic approach, the peptide/proteins in the fluid and verify possible variations occurring during the exfoliating process. The results obtained confirmed the presence in GCF of α-Defensins 1-4, Thymosin β4 and Thymosin β10, as described in previous works and revealed the presence of other interesting peptides never described before in GCF such as specific fragments of α-1-antitrypsin, α-1-antichymotrypsin; fragments of Thymosin β4 and Thymosin β10; Fibrinopeptide A and its fragments and Fibrinopeptide B; S100A8 and S100A9, LVV Hemorphin-7 (hemoglobin chain β fragment), as well as some other peptides deriving from α and β subunits of hemoglobin. Statistical analysis evidenced different levels in 5 proteins/peptides in the three groups. Our study demonstrate that an in-depth analysis of a biological fluid like GCF, present in small amount, can provide useful information for the understanding of different biological processes like teeth eruption. Data are available via ProteomeXchange with identifier PXD016010 and PXD016049. SIGNIFICANCE: GCF due to his site-specific nature has a great potential in containing factors that are specific for action at a given site and might have diagnostic value to detect qualitative and quantitative variations of proteins/peptides composition linked to physiological or pathological conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jprot.2020.103890DOI Listing
August 2020

Multiple Herpes Simplex Virus-1 (HSV-1) Reactivations Induce Protein Oxidative Damage in Mouse Brain: Novel Mechanisms for Alzheimer's Disease Progression.

Microorganisms 2020 Jun 29;8(7). Epub 2020 Jun 29.

Department of Public Health and Infectious Diseases, Sapienza University of Rome, Laboratory affiliated to Istituto Pasteur Italia-Fondazione Cenci Bolognetti, 00185 Rome, Italy.

Compelling evidence supports the role of oxidative stress in Alzheimer's disease (AD) pathophysiology. Interestingly, Herpes simplex virus-1 (HSV-1), a neurotropic virus that establishes a lifelong latent infection in the trigeminal ganglion followed by periodic reactivations, has been reportedly linked both to AD and to oxidative stress conditions. Herein, we analyzed, through biochemical and redox proteomic approaches, the mouse model of recurrent HSV-1 infection we previously set up, to investigate whether multiple virus reactivations induced oxidative stress in the mouse brain and affected protein function and related intracellular pathways. Following multiple HSV-1 reactivations, we found in mouse brains increased levels of oxidative stress hallmarks, including 4-hydroxynonenal (HNE), and 13 HNE-modified proteins whose levels were found significantly altered in the cortex of HSV-1-infected mice compared to controls. We focused on two proteins previously linked to AD pathogenesis, i.e., glucose-regulated protein 78 (GRP78) and collapsin response-mediated protein 2 (CRMP2), which are involved in the unfolded protein response (UPR) and in microtubule stabilization, respectively. We found that recurrent HSV-1 infection disables GRP78 function and activates the UPR, whereas it prevents CRMP2 function in mouse brains. Overall, these data suggest that repeated HSV-1 reactivation into the brain may contribute to neurodegeneration also through oxidative damage.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/microorganisms8070972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7409037PMC
June 2020

Top-Down Proteomics of Human Saliva Discloses Significant Variations of the Protein Profile in Patients with Mastocytosis.

J Proteome Res 2020 08 6;19(8):3238-3253. Epub 2020 Jul 6.

Dipartimento di Scienze della Vita e dell'Ambiente, Università di Cagliari, 09124 Cagliari, Italy.

Mastocytosis is a myeloproliferative neoplasm causing abnormal clonal mast cell accumulation in different tissues, such as skin and bone marrow. A cutaneous subtype (CM) is distinguished from a systemic one (SM); SM patients can be grouped into SM with (SM+C) or without (SM-C) additional cutaneous lesions, and their classification is often challenging. This study was purposed to highlight variations in the salivary proteome of patients with different mastocytosis subtypes and compared to healthy controls. A top-down proteomics approach coupled to a label-free quantitation revealed salivary profiles in patients different from those of controls and a down-regulation of peptides/proteins involved in the mouth homeostasis and defense, such as statherin, histatins, and acidic proline-rich proteins (aPRPs), and in innate immunity and inflammation, such as the cathepsin inhibitors, suggesting a systemic condition associated with an exacerbated inflammatory state. The up-regulation of antileukoproteinase and S100A8 suggested a protective role against the disease status. The two SM forms were distinguished by the lower levels of truncated forms of aPRPs, statherin, P-B peptide, and cystatin D and the higher levels of thymosin β4 and α-defensins 1 and 4 in SM-C patients with respect to SM+C. Data are available via ProteomeXchange with identifier PXD017759.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jproteome.0c00207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8008451PMC
August 2020

Proteomic Analysis of the Acid-Insoluble Fraction of Whole Saliva from Patients Affected by Different Forms of Non-histaminergic Angioedema.

J Clin Immunol 2020 08 10;40(6):840-850. Epub 2020 Jun 10.

Dept of Life and Environmental Sciences, University of Cagliari, 09042, Monserrato, CA, Italy.

We analyzed by bidimensional electrophoresis the acid-insoluble fraction of saliva from three classes of angioedema patients and a healthy control group, highlighting significant variations of several normalized spot volumes. Characterization of the corresponding proteins was performed by in-gel tryptic digestion of the spots, followed by high-resolution HPLC-ESI-MS/MS analysis of tryptic mixtures. By this strategy, 16 differentially-expressed proteins among two or more groups were identified. We found higher concentration of proteins involved in immune response (interleukin-1 receptor antagonist and annexin A1), and of moonlighting proteins acting as plasminogen receptors (glyceraldehyde-3-phosphate dehydrogenase, α-enolase, and annexin A2) in patients affected by the idiopathic non-histaminergic or hereditary angioedema with unknown origin with respect to healthy controls. These data provide new information on the molecular basis of these less characterized types of angioedema. Graphical Abstract Graphical Abstract.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10875-020-00802-wDOI Listing
August 2020

Ultra-rapid glutathionylation of chymotrypsinogen in its molten globule-like conformation: A comparison to archaeal proteins.

Sci Rep 2020 06 2;10(1):8943. Epub 2020 Jun 2.

Dipartimento di Scienze e Tecnologie Chimiche, Università di Roma "Tor Vergata", Rome, Italy.

Chymotrypsinogen, when reduced and taken to its molten globule-like conformation, displays a single cysteine with an unusual kinetic propensity toward oxidized glutathione (GSSG) and other organic thiol reagents. A single residue, identified by mass spectrometry like Cys1, reacts with GSSG about 1400 times faster than an unperturbed protein cysteine. A reversible protein-GSSG complex and a low pK (8.1 ± 0.1) make possible such astonishing kinetic property which is absent toward other natural disulfides like cystine, homocystine and cystamine. An evident hyper-reactivity toward 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and 1-chloro-2,4-dinitrobenzene (CDNB) was also found for this specific residue. The extraordinary reactivity toward GSSG is absent in two proteins of the thermophilic archaeon Sulfolobus solfataricus, an organism lacking glutathione: the Protein Disulphide Oxidoreductase (SsPDO) and the Bacterioferritin Comigratory Protein 1 (Bcp1) that displays Cys residues with an even lower pK value (7.5 ± 0.1) compared to chymotrypsinogen. This study, which also uses single mutants in Cys residues for Bcp1, proposes that this hyper-reactivity of a single cysteine, similar to that found in serum albumin, lysozyme, ribonuclease, may have relevance to drive the "incipit" of the oxidative folding of proteins from organisms where the glutathione/oxidized glutathione (GSH/GSSG) system is present.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-65696-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265447PMC
June 2020

Exploring the HeLa Dark Mitochondrial Proteome.

Front Cell Dev Biol 2020 5;8:137. Epub 2020 Mar 5.

Proteomics and Metabonomics Unit, IRCCS-Fondazione Santa Lucia, Rome, Italy.

In the framework of the Human Proteome Project initiative, we aim to improve mapping and characterization of mitochondrial proteome. In this work we implemented an experimental workflow, combining classical biochemical enrichments and mass spectrometry, to pursue a much deeper definition of mitochondrial proteome and possibly mine mitochondrial uncharacterized . We fractionated in two compartments mitochondria enriched from HeLa cells in order to annotate 4230 proteins in both fraction by means of a multiple-enzyme digestion (trypsin, chymotrypsin and Glu-C) followed by mass spectrometry analysis using a combination of Data Dependent Acquisition (DDA) and Data Independent Acquisition (DIA). We detected 22 mitochondrial dark proteins not annotated for their function and we provide their relative abundance inside the mitochondrial organelle. Considering this work as a pilot study we expect that the same approach, in different biological system, could represent an advancement in the characterization of the human mitochondrial proteome providing uncharted ground to explore the mitonuclear phenotypic relationships. All spectra have been deposited to ProteomeXchange with PXD014201 and PXD014200 identifier.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fcell.2020.00137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7066081PMC
March 2020

Protein Oxidative Damage in UV-Related Skin Cancer and Dysplastic Lesions Contributes to Neoplastic Promotion and Progression.

Cancers (Basel) 2020 01 1;12(1). Epub 2020 Jan 1.

Department of RiDAIT, the Regina Elena National Cancer Institute IRCCS, via Elio Chianesi 53, 00144 Rome, Italy.

The ultraviolet (UV) component of solar radiation is the major driving force of skin carcinogenesis. Most of studies on UV carcinogenesis actually focus on DNA damage while their proteome-damaging ability and its contribution to skin carcinogenesis have remained largely underexplored. A redox proteomic analysis of oxidized proteins in solar-induced neoplastic skin lesion and perilesional areas has been conducted showing that the protein oxidative burden mostly concerns a selected number of proteins participating to a defined set of functions, namely: chaperoning and stress response; protein folding/refolding and protein quality control; proteasomal function; DNA damage repair; protein- and vesicle-trafficking; cell architecture, adhesion/extra-cellular matrix (ECM) interaction; proliferation/oncosuppression; apoptosis/survival, all of them ultimately concurring either to structural damage repair or to damage detoxication and stress response. In peri-neoplastic areas the oxidative alterations are conducive to the persistence of genetic alterations, dysfunctional apoptosis surveillance, and a disrupted extracellular environment, thus creating the condition for transformant clones to establish, expand and progress. A comparatively lower burden of oxidative damage is observed in neoplastic areas. Such a finding can reflect an adaptive selection of best fitting clones to the sharply pro-oxidant neoplastic environment. In this context the DNA damage response appears severely perturbed, thus sustaining an increased genomic instability and an accelerated rate of neoplastic evolution. In conclusion UV radiation, in addition to being a cancer-initiating agent, can act, through protein oxidation, as a cancer-promoting agent and as an inducer of genomic instability concurring with the neoplastic progression of established lesions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers12010110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7017152PMC
January 2020

Mapping of Transglutaminase-2 Sites of Human Salivary Small Basic Proline-Rich Proteins by HPLC-High-Resolution ESI-MS/MS.

J Proteome Res 2020 01 6;19(1):300-313. Epub 2019 Nov 6.

Department of Life and Environmental Sciences , University of Cagliari, Cittadella Univ. Monserrato , Monserrato, Cagliari 09042 , Italy.

Because of the distinctive features of the oral cavity, the determination of the proteins involved in the formation of the "oral protein pellicle" is demanding. The present study investigated the susceptibility of several human basic proline-rich peptides, named P-H, P-D, P-F, P-J, and II-2, as substrates of transglutaminase-2. The reactivity of the P-C peptide and statherin was also investigated. Peptides purified from human whole saliva were incubated with the enzyme in the presence or in the absence of monodansyl-cadaverine. Mass spectrometry analyses of the reaction products highlighted that P-H and P-D (P and A variants) were active substrates, II-2 was less reactive, and P-F and P-J showed very low reactivity. P-C and statherin were highly reactive. All of the peptides formed cyclo derivatives, and only specific glutamine residues were involved in the cycle formation and reacted with monodansyl-cadaverine: Q of P-H, Q of P-D, Q of II-2, Q of P-C, and Q of statherin were the principal reactive residues. One or two secondary glutamine residues of only P-H, P-D P, P-C, and statherin were hierarchically susceptible to the reaction with monodansyl-cadaverine. MS and MS/MS data were deposited to the ProteomeXchange Consortium ( http://www.ebi.ac.uk/pride ) via the PRIDE partner repository with the data set identifier PXD014658.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jproteome.9b00527DOI Listing
January 2020

Enrichments of post-translational modifications in proteomic studies.

J Sep Sci 2020 Jan 6;43(1):313-336. Epub 2019 Nov 6.

Dipartimento di Scienze della Vita e dell'Ambiente, Università di Cagliari, Cagliari, Italy.

More than 300 different protein post-translational modifications are currently known, but only a few have been extensively investigated because modified proteoforms are commonly present in sub-stoichiometry amount. For this reason, improvement of specific enrichment techniques is particularly useful for the proteomic characterization of post-translationally modified proteins. Enrichment proteomic strategies could help the researcher in the challenging issue to decipher the complex molecular cross-talk existing between the different factors influencing the cellular pathways. In this review the state of art of the platforms applied for the enrichment of specific and most common post-translational modifications, such as glycosylation and glycation, phosphorylation, sulfation, redox modifications (i.e. sulfydration and nitrosylation), methylation, acetylation, and ubiquitinylation, are described. Enrichments strategies applied to characterize less studied post-translational modifications are also briefly discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jssc.201900804DOI Listing
January 2020

Zimmermann-Laband-1 Syndrome: Clinical, Histological, and Proteomic Findings of a 3-Year-Old Patient with Hereditary Gingival Fibromatosis.

Biomedicines 2019 Jun 29;7(3). Epub 2019 Jun 29.

Istituto di Odontoiatria, Fondazione Policlinico Universitario A. Gemelli IRCCS, Università Cattolica del Sacro Cuore, 00168 Rome, Italy.

Background: Zimmermann-Laband-1 syndrome (ZLS-1; OMIM# 135500) is a rare genetic disorder whose oral pathognomonic sign is the development of progressive, diffuse, and severe gingival hypertrophy. Most children with abnormally gingival hyperplasia may also present multiple unerupted teeth and skeletal deformities of maxillary arches (i.e., skeletal anterior open bite). Despite phenotypic variability of the clinical spectrum, gingival fibromatosis is the hallmark of ZLS-1.

Method: In this study, we report a 3-year-old male patient with a ZLS-1-related gingival overgrowth and failure of eruption of the deciduous teeth in the molar area. Surgical excision was performed under general anesthesia.

Results: At three weeks follow-up, esthetics was significantly improved in terms of gingival appearance, and teeth eruption allowed an adequate masticatory function.

Conclusion: In severe cases, surgical removal of the hyperplasic fibrous tissue may be required to expose unerupted teeth and establish a proper gingival contour. Surgical excision under general anesthesia is an elective procedure for patients with special needs, mental disability, as well as young and adult patients with dental anxiety type II and IV associated with poor oral health.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/biomedicines7030048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6783959PMC
June 2019

Investigating the Protein Signature of Adamantinomatous Craniopharyngioma Pediatric Brain Tumor Tissue: Towards the Comprehension of Its Aggressive Behavior.

Dis Markers 2019 2;2019:3609789. Epub 2019 May 2.

Istituto di Chimica del Riconoscimento Molecolare, Consiglio Nazionale delle Ricerche, Roma, Italy.

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

Download full-text PDF

Source
http://dx.doi.org/10.1155/2019/3609789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6525946PMC
December 2019

Restoration of aberrant mTOR signaling by intranasal rapamycin reduces oxidative damage: Focus on HNE-modified proteins in a mouse model of down syndrome.

Redox Biol 2019 05 9;23:101162. Epub 2019 Mar 9.

Department of Biochemical Sciences, Sapienza University of Rome, Rome, Italy. Electronic address:

Increasing evidences support the notion that the impairment of intracellular degradative machinery is responsible for the accumulation of oxidized/misfolded proteins that ultimately results in the deposition of protein aggregates. These events are key pathological aspects of "protein misfolding diseases", including Alzheimer disease (AD). Interestingly, Down syndrome (DS) neuropathology shares many features with AD, such as the deposition of both amyloid plaques and neurofibrillary tangles. Studies from our group and others demonstrated, in DS brain, the dysfunction of both proteasome and autophagy degradative systems, coupled with increased oxidative damage. Further, we observed the aberrant increase of mTOR signaling and of its down-stream pathways in both DS brain and in Ts65Dn mice. Based on these findings, we support the ability of intranasal rapamycin treatment (InRapa) to restore mTOR pathway but also to restrain oxidative stress resulting in the decreased accumulation of lipoxidized proteins. By proteomics approach, we were able to identify specific proteins that showed decreased levels of HNE-modification after InRapa treatment compared with vehicle group. Among MS-identified proteins, we found that reduced oxidation of arginase-1 (ARG-1) and protein phosphatase 2A (PP2A) might play a key role in reducing brain damage associated with synaptic transmission failure and tau hyperphosphorylation. InRapa treatment, by reducing ARG-1 protein-bound HNE levels, rescues its enzyme activity and conceivably contribute to the recovery of arginase-regulated functions. Further, it was shown that PP2A inhibition induces tau hyperphosphorylation and spatial memory deficits. Our data suggest that InRapa was able to rescue PP2A activity as suggested by reduced p-tau levels. In summary, considering that mTOR pathway is a central hub of multiple intracellular signaling, we propose that InRapa treatment is able to lower the lipoxidation-mediated damage to proteins, thus representing a valuable therapeutic strategy to reduce the early development of AD pathology in DS population.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.redox.2019.101162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6859577PMC
May 2019

Oxidative Stress and Bronchopulmonary Dysplasia: Evidences From Microbiomics, Metabolomics, and Proteomics.

Front Pediatr 2019 13;7:30. Epub 2019 Feb 13.

Neonatal Intensive Care Unit, Neonatal Pathology and Neonatal Section, Azienda Ospedaliero-Universitaria Cagliari and University of Cagliari, Cagliari, Italy.

Bronchopulmonary dysplasia is a major issue affecting morbidity and mortality of surviving premature babies. Preterm newborns are particularly susceptible to oxidative stress and infants with bronchopulmonary dysplasia have a typical oxidation pattern in the early stages of this disease, suggesting the important role of oxidative stress in its pathogenesis. Bronchopulmonary dysplasia is a complex disease where knowledge advances as new investigative tools become available. The explosion of the "omics" disciplines has recently affected BPD research. This review focuses on the new evidence coming from microbiomics, metabolomics and proteomics in relation to oxidative stress and pathogenesis of bronchopulmonary dysplasia. Since the pathogenesis is not yet completely understood, information gained in this regard would be important for planning an efficacious prevention and treatment strategy for the future.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fped.2019.00030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6381008PMC
February 2019

Protein nitration profile of CD3 lymphocytes from Alzheimer disease patients: Novel hints on immunosenescence and biomarker detection.

Free Radic Biol Med 2018 12 12;129:430-439. Epub 2018 Oct 12.

Department of Biomedical Sciences and Biotechnologies, University of Brescia, Brescia, Italy.

Alzheimer's disease (AD) is a progressive form of dementia characterized by increased production of amyloid-β plaques and hyperphosphorylated tau protein, mitochondrial dysfunction, elevated oxidative stress, reduced protein clearance, among other. Several studies showed systemic modifications of immune and inflammatory systems due, in part, to decreased levels of CD3 lymphocytes in peripheral blood in AD. Considering that oxidative stress, both in the brain and in the periphery, can influence the activation and differentiation of T-cells, we investigated the 3-nitrotyrosine (3-NT) proteome of blood T-cells derived from AD patients compared to non-demented (ND) subjects by using a proteomic approach. 3-NT is a formal protein oxidation and index of nitrosative stress. We identified ten proteins showing increasing levels of 3-NT in CD3 T-cells from AD patients compared with ND subjects. These proteins are involved in energy metabolism, cytoskeletal structure, intracellular signaling, protein folding and turnover, and antioxidant response and provide new insights into the molecular mechanism that impact reduced T-cell differentiation in AD. Our results highlight the role of peripheral oxidative stress in T-cells related to immune-senescence during AD pathology focusing on the specific targets of protein nitration that conceivably can be suitable to further therapies. Further, our data demonstrate common targets of protein nitration between the brain and the periphery, supporting their significance as disease biomarkers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.freeradbiomed.2018.10.414DOI Listing
December 2018

Top-down proteomic profiling of human saliva in multiple sclerosis patients.

J Proteomics 2018 09 4;187:212-222. Epub 2018 Aug 4.

Department of Life and Environmental Sciences, Biomedical Section, University of Cagliari, Monserrato Campus, 09042 Monserrato, Cagliari, Italy.

Multiple sclerosis is a chronic disease of the central nervous system characterized by inflammation, demyelination and neurodegeneration which is of undetermined origin. To date a single diagnostic test of multiple sclerosis does not exists and novel biomarkers are demanded for a more accurate and early diagnosis. In this study, we performed the quantitative analysis of 119 salivary peptides/proteins from 49 multiple sclerosis patients and 54 healthy controls by a mass spectrometry-based top-down proteomic approach. Statistical analysis evidenced different levels on 23 proteins: 8 proteins showed lower levels in multiple sclerosis patients with respect to controls and they were mono- and di-oxidized cystatin SN, mono- and di-oxidized cystatin S1, mono-oxidized cystatin SA and mono-phosphorylated statherin. 15 proteins showed higher levels in multiple sclerosis patients with respect to controls and they were antileukoproteinase, two proteoforms of Prolactin-Inducible Protein, P-C peptide (Fr.1-14, Fr. 26-44, and Fr. 36-44), SV1 fragment of statherin, cystatin SN Des, cystatin SN P → L variant, and cystatin A T → M variant. The differences observed between the salivary proteomic profile of patients suffering from multiple sclerosis and healthy subjects is consistent with the inflammatory condition and altered immune response typical of the pathology. Data are available via ProteomeXchange with identifier PXD009440.

Significance: To date a single diagnostic test of multiple sclerosis does not exist, and diagnosis is based on multiple tests which mainly include the analysis of cerebrospinal fluid. However, the need for lumbar puncture makes the analysis of cerebrospinal fluid impractical for monitoring disease activity and response to treatment. The possible use of saliva as a diagnostic fluid for oral and systemic diseases has been largely investigated, but only marginally in multiple sclerosis compared to other body fluids. Our study demonstrates that the salivary proteome of multiple sclerosis patients differs considerably compared to that of sex and age matched healthy individuals and suggests that some differences might be associated with the different disease-modifying therapy used to treat multiple sclerosis patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jprot.2018.07.019DOI Listing
September 2018

Extensive Characterization of the Human Salivary Basic Proline-Rich Protein Family by Top-Down Mass Spectrometry.

J Proteome Res 2018 09 20;17(9):3292-3307. Epub 2018 Aug 20.

Department of Life and Environmental Sciences , University of Cagliari, Cittadella Univ. Monserrato , Monserrato 09042 , Cagliari , Italy.

Human basic proline-rich proteins and basic glycosylated proline-rich proteins, encoded by the polymorphic PRB1-4 genes and expressed only in parotid glands, are the most complex family of adult salivary proteins. The family includes 11 parent peptides/proteins and more than 6 parent glycosylated proteins, but a high number of proteoforms with rather similar structures derive from polymorphisms and post-translational modifications. 55 new components of the family were characterized by top-down liquid chromatography-mass spectrometry and tandem-mass platforms, bringing the total number of proteoforms to 109. The new components comprise the three variants P-H S → A, P-Ko P → S, and P-Ko A → S and several of their naturally occurring proteolytic fragments. The paper represents an updated reference for the peptides included in the heterogeneous family of proteins encoded by PRB1/PRB4. MS data are available via ProteomeXchange with the identifier PXD009813.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jproteome.8b00444DOI Listing
September 2018

Thymosin fraction 5 re-evaluated after 35 years by high-resolution mass spectrometry.

Expert Opin Biol Ther 2018 07;18(sup1):199-203

a Friedrich-Alexander-University Erlangen-Nuremberg , Institute of Biochemistry , Erlangen , Germany.

Objectives: We reevaluated a lyophilized sample of thymosin fraction 5, stored for 37 years at room temperature, by high-resolution mass spectrometry in terms of stability and yet uncharacterized polypeptides that could be biological important substances.

Methods: A top-down proteomic platform based on high-performance liquid chromatography (HPLC) coupled to high-resolution LTQ-Orbitrap mass spectrometry (MS) was applied to molecular characterization of polypeptides present in thymosin fraction 5.

Results: We detected more than 100 monoisotopic masses corresponding to thymosin β4 and truncated forms of ubiquitin, prothymosin α, thymosin β4, and thymosin β9. Additionally, we discovered a new polypeptide present in thymosin fraction 5 and identified it as intact SH3 domain-binding glutamic acid-rich-like protein 3.

Conclusion: In spite of the well-known proteolytic processes inherent to the preparation of thymosin fraction 5, still uncharacterized polypeptides as well as truncated forms of already well-known thymosins are present in fraction 5 after long-term storage. Therefore, continuing characterization of thymosin fraction 5 is even nowadays highly promising.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/14712598.2018.1474196DOI Listing
July 2018

Proteomic identification of altered protein O-GlcNAcylation in a triple transgenic mouse model of Alzheimer's disease.

Biochim Biophys Acta Mol Basis Dis 2018 10 18;1864(10):3309-3321. Epub 2018 Jul 18.

Department of Biochemical Sciences "A. Rossi Fanelli", Sapienza University of Rome, Rome, Italy. Electronic address:

PET scan analysis demonstrated the early reduction of cerebral glucose metabolism in Alzheimer disease (AD) patients that can make neurons vulnerable to damage via the alteration of the hexosamine biosynthetic pathway (HBP). Defective HBP leads to flawed protein O-GlcNAcylation coupled, by a mutual inverse relationship, with increased protein phosphorylation on Ser/Thr residues. Altered O-GlcNAcylation of Tau and APP have been reported in AD and is closely related with pathology onset and progression. In addition, type 2 diabetes patients show an altered O-GlcNAcylation/phosphorylation that might represent a link between metabolic defects and AD progression. Our study aimed to decipher the specific protein targets of altered O-GlcNAcylation in brain of 12-month-old 3×Tg-AD mice compared with age-matched non-Tg mice. Hence, we analysed the global O-GlcNAc levels, the levels and activity of OGT and OGA, the enzymes controlling its cycling and protein specific O-GlcNAc levels using a bi-dimensional electrophoresis (2DE) approach. Our data demonstrate the alteration of OGT and OGA activation coupled with the decrease of total O-GlcNAcylation levels. Data from proteomics analysis led to the identification of several proteins with reduced O-GlcNAcylation levels, which belong to key pathways involved in the progression of AD such as neuronal structure, protein degradation and glucose metabolism. In parallel, we analysed the O-GlcNAcylation/phosphorylation ratio of IRS1 and AKT, whose alterations may contribute to insulin resistance and reduced glucose uptake. Our findings may contribute to better understand the role of altered protein O-GlcNAcylation profile in AD, by possibly identifying novel mechanisms of disease progression related to glucose hypometabolism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbadis.2018.07.017DOI Listing
October 2018

Cryptides: latent peptides everywhere.

Crit Rev Biochem Mol Biol 2018 06 22;53(3):246-263. Epub 2018 Mar 22.

c Dipartimento di Scienze della Vita e dell'Ambiente , Università di Cagliari , Cagliari , Italy.

Proteomic surveys with top-down platforms are today revealing thousands of naturally occurring fragments of bigger proteins. Some of them have not functional meaning because they derive from pathways responsible for protein degradation, but many have specific functions, often completely different from that one of the parent proteins. These peptides encrypted in the protein sequence are nowadays called cryptides. They are frequent in the animal and plant kingdoms and represent a new interesting -omic field of investigation. To point out how much widespread is their presence, we describe here the most studied cryptides from very common sources such as serum albumin, immunoglobulins, hemoglobin, and from saliva and milk proteins. Given its vastness, it is unfeasible to cover the topic exhaustively, therefore only several selected examples of cryptides from other sources are thereafter reported. Demanding is the development of new -omic platforms for the functional screening of new cryptides, which could provide suggestion for peptides and peptido-mimetics with variegate fields of application.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/10409238.2018.1447543DOI Listing
June 2018

Marked Differences in the Submandibular Salivary Proteome between Sardinian Alcohol-Preferring and Sardinian Alcohol-Non Preferring Rats Revealed by an Integrated Top-Down-Bottom-Up Proteomic Platform.

J Proteome Res 2018 01 16;17(1):455-469. Epub 2017 Nov 16.

Istituto di Biochimica e Biochimica Clinica, Facoltà di Medicina, Università Cattolica , Largo Francesco Vito 1 00168, Rome, Italy.

Sardinian alcohol-preferring (sP) and Sardinian alcohol-non preferring (sNP) rats have been selectively bred for opposite alcohol preference and consumption. Aiming to verify possible differences at the proteomics level between sP and sNP rats, we investigated the salivary proteome by a a liquid chromatography-mass spectrometry top-down-bottom-up integrated approach. For this purpose, submandibular saliva was collected from alcohol-naive sP and sNP rats under isoprenaline stimulation. A total of 200 peptides and proteins were detected and quantified in the two rat lines, 149 of which were characterized in their naturally occurring structure. The data are available via ProteomeXchange with identifier PXD006997. Surprisingly, sP rats exhibited marked quantitative and qualitative differences with respect to sNP rats, namely higher levels of proteoforms originating from submandibular gland protein C, and from submandibular rat protein 2, as well as those of several unidentified peptides and proteins. sP rats expressed some proteins not detectable in sNP rats such as the glutamine and glutamic acid-rich protein (GRP)-CB. The isoform GRP-B, detectable in both rat lines, was more abundant in sNP rats. The submandibular saliva of sNP rats was also characterized by very high levels of GRP-B proteolytic peptides and rat salivary protein 1. Whether these differences could contribute to the opposite alcohol preference and consumption of sP and sNP rats is currently unknown and requires further investigation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jproteome.7b00632DOI Listing
January 2018

Salivary Cystatins: Exploring New Post-Translational Modifications and Polymorphisms by Top-Down High-Resolution Mass Spectrometry.

J Proteome Res 2017 11;16(11):4196-4207

Department of Life and Environmental Sciences, Biomedical Section, University of Cagliari , Monserrato Campus, 09042 Monserrato, Cagliari, Italy.

Cystatins are a complex family of cysteine peptidase inhibitors. In the present study, various proteoforms of cystatin A, cystatin B, cystatin S, cystatin SN, and cystatin SA were detected in the acid-soluble fraction of human saliva and characterized by a top-down HPLC-ESI-MS approach. Proteoforms of cystatin D were also detected and characterized by an integrated top-down and bottom-up strategy. The proteoforms derive from coding sequence polymorphisms and post-translational modifications, in particular, phosphorylation, N-terminal processing, and oxidation. This study increases the current knowledge of salivary cystatin proteoforms and provides the basis to evaluate possible qualitative/quantitative variations of these proteoforms in different pathological states and reveal new potential salivary biomarkers of disease. Data are available via ProteomeXchange with identifier PXD007170.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jproteome.7b00567DOI Listing
November 2017

Top-down HPLC-ESI-MS proteomic analysis of saliva of edentulous subjects evidenced high levels of cystatin A, cystatin B and SPRR3.

Arch Oral Biol 2017 May 31;77:68-74. Epub 2017 Jan 31.

Dipartimento di Scienze della Vita e dell'Ambiente, Università di Cagliari, 09042, Monserrato CA, Italy.

Objective: This study aims to analyze the salivary peptidome/proteome of edentulous subject with respect to dentate control subjects.

Design: Unstimulated whole saliva, collected from 11 edentulous subjects (age 60-76 years) and 11 dentate age-matched control subjects, was immediately treated with 0.2% aqueous trifluoroacetic acid and the acidic soluble fraction analyzed by High Performace Liquid Chromatography-Mass Spectrometry. The relative abundance of the salivary peptides/proteins was determined by measuring the area of the High Performace Liquid Chromatography-Mass Spectrometry eXtracted Ion Current peaks which is linearly proportional to peptide/protein concentration under identical experimental conditions. Levels of salivary peptides/proteins in the two groups were compared by the nonparametric Mann-Whitney test to evidence statistically significant differences.

Results: Levels of cystatin A, S-glutathionylated, S-cystenylated, S-S dimer derivatives of cystatin B and S-glutathionylated derivative of SPRR3, were found significantly higher in edentulous subjects with respect to dentate controls. The major peptides and proteins typically deriving from salivary glands did not show any statistically significant differences.

Conclusions: Cystatin A, S-glutathionylated, S-cystenylated, S-S dimer derivatives of cystatin B and S-glutathionylated derivative of SPRR3, which are mainly of intracellular origin and represent the major constituents of the cornified cell envelope are a clue of inflammation of mucosal epithelia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.archoralbio.2017.01.021DOI Listing
May 2017

The extreme hyper-reactivity of selected cysteines drives hierarchical disulfide bond formation in serum albumin.

FEBS J 2016 11 26;283(22):4113-4127. Epub 2016 Oct 26.

Department of Chemical Sciences and Technologies, University of Rome 'Tor Vergata', Italy.

After mild reduction of serum albumin, seven among the 34 cysteines forming the disulfide network displayed a surprising hyper-reactivity. Compared to the thiol group of glutathione, the average reactivity of these cysteines towards disulfides and thiol reagents was more than 100 times higher. Using mass spectrometry and kinetic data, we identified all these unusual residues, with Cys75, Cys123 and Cys264 showing the highest reactivity. This effect was mainly due to a low pK of the sulfhydryl groups and may explain the very fast formation of early disulfides in the nascent protein suggesting the existence of a hierarchical propensity to form such covalent links in selected regions during oxidative folding. An identical pattern of hyper-reactive cysteines was found in albumins from six different mammals. This hyper-reactivity is much higher than the one found in other proteins containing multiple cysteines only devoted to structural disulfide bonds. It is possible that such hyper-reactive cysteines could also be present in other proteins, although their existence has been completely ignored so far.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/febs.13909DOI Listing
November 2016

Thymosin β and β in Sjögren's syndrome: saliva proteomics and minor salivary glands expression.

Arthritis Res Ther 2016 10 6;18(1):229. Epub 2016 Oct 6.

Division of Rheumatology, Fondazione Policlinico Universitario Agostino Gemelli, Rome, Italy.

Background: In the present study, we investigated whether thymosin β (Tβ) in saliva and in minor salivary glands is differentially expressed in patients with primary Sjögren's syndrome (pSS) and patients with autoimmune diseases (systemic sclerosis [SSc], systemic lupus erythematosus [SLE], and rheumatoid arthritis [RA], with and without sicca syndrome [ss]).

Methods: Saliva specimens of nine patients with pSS, seven with ss/SSc, seven with ss/SLE, seven with ss/RA, seven with SSc, seven with SLE, and seven with RA, as well as ten healthy subjects, were analyzed using a high-performance liquid chromatograph coupled with a mass spectrometer equipped with an electrospray ionization source to investigate the presence and levels of Tβ, Tβ sulfoxide, and Tβ. Immunostaining for Tβ and Tβ was performed on minor salivary glands of patients with pSS and ss.

Results: Tβ levels were statistically higher in patients with pSS with respect to the other subgroups. Tβ was detectable in 66.7 % of patients with pSS and in 42.8 % of those with ss/SSc, while Tβ sulfoxide was detectable in 44.4 % of patients with pSS and in 42.9 % of those with ss/SSc. Tβ and Tβ sulfoxide were not detectable in patients without associated ss and in healthy control subjects. Regarding thymosin immunostaining, all patients had immunoreactivity for Tβ, and a comparable distribution pattern in the four different subgroups of patients was observed. Tβ immunoreactivity was present in patients with ss/SSc and those with ss/SLE, while it was completely absent in patients with pSS and those with ss/RA.

Conclusions: Our data show that higher salivary Tβ expression characterizes patients with pSS, while Tβ sulfoxide and Tβ salivary expression was selectively present in patients with sicca symptoms. Moreover, at the immunohistochemical level in patients with pSS, minor salivary glands showed a peculiar pattern characterized by immunostaining for Tβ in acinar cells in the absence of any reactivity for Tβ. These findings, taken together, suggest a different role for Tβ and Tβ in patients with pSS who have ss and other autoimmune disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13075-016-1134-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5053072PMC
October 2016

Proteomics applied to pediatric medicine: opportunities and challenges.

Expert Rev Proteomics 2016 09 25;13(9):883-94. Epub 2016 Aug 25.

b Istituto di Chimica del Riconoscimento Molecolare - UOS Roma , Consiglio Nazionale delle Ricerche , Rome , Italy.

Introduction: Care in pediatrics often refers to treatments directed to adults. However, childhood is a specific life period, with molecular pathways connected to development and thereby it requires distinctive considerations and special treatments under disease. Proteomics can help to elucidate the molecular mechanisms underlying the human development and disease onset in pediatric age and this review is devoted to underline the results recently obtained in the field.

Areas Covered: The contribution of proteomics to the characterization of physiological modifications occurring during human development is presented. The proteomic studies carried out to elucidate the molecular mechanisms underlying different pediatric pathologies and to discover new markers for early diagnosis and prognosis of disease, comprising genetic and systemic pathologies, sepsis and pediatric oncology are thereafter reported. The investigations concerning milk composition in human and farm mammals are also presented. Finally, the chances offered by the integration of different -omic platforms are discussed. Expert commentary: The growing utilization of holistic technologies such as proteomics, metabolomics and microbiomics will allow, in the near future, to define at the molecular level the complexity of human development and related diseases, with great benefit for future generations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/14789450.2016.1221764DOI Listing
September 2016

Cell wall composition and biofilm formation of azoles-susceptible and -resistant Candida glabrata strains.

J Chemother 2017 Jun 20;29(3):164-172. Epub 2016 Jul 20.

b Department of Public Health and Infectious Diseases , "Sapienza" University of Rome , P.le Aldo Moro 5, Rome 00161 , Italy.

In the present study, three strains of Candida glabrata have been investigated to shed light on the mechanisms involved in azole resistance during adherence and biofilm formation. In particular, a clinical isolate, susceptible to azole-based drugs, DSY562 and two different resistant mutagenic strains deriving from DSY562, SFY114 and SFY115, have been analysed with different approaches for their cell wall composition and properties. A proteomic analysis revealed that the expression of six cell wall-related proteins and biofilm formation varied between the strains. The SFY114 and SFY115 strains resulted to be less hydrophobic than the susceptible parental counterpart DSY562, on the other hand they showed a higher amount in total cell wall polysaccharides fraction in the total cell wall. Accordingly to the results obtained from the hydrophobicity and adherence assays, in the resistant strain SFY115 the biofilm formation decreased compared to the parental strain DSY562. Finally, the total glucose amount in resistant SFY115 was about halved in comparison to other strains. Taken together all these data suggest that azole drugs may affect the cell wall composition of C. glabrata, in relation to the different pathogenic behaviours.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/1120009X.2016.1199507DOI Listing
June 2017

Proteomic characterization of the acid-insoluble fraction of whole saliva from preterm human newborns.

J Proteomics 2016 09 16;146:48-57. Epub 2016 Jun 16.

Department of Life and Environmental Sciences, Biomedical Section, University of Cagliari, Monserrato Campus, 09042 Monserrato, CA, Italy. Electronic address:

The acid-insoluble salivary proteome obtained by addition of TFA to whole human saliva from adults, preterm and at-term newborns has been analysed by 2-DE in order to evidence differences among the three groups, and integrate data previously obtained on the acid-soluble fraction. 2-DE spots differentially expressed among the three groups were submitted to in-gel tryptic digestion and the peptide mixtures analysed by high resolution HPLC–ESI–MS/MS. By this strategy, we identified 3 over-expressed proteins in at-term newborns with respect to preterm newborns and adults (BPI fold-containing family A member 1, annexin A1, and keratin type 1 cytoskeletal 13), and several over-expressed proteins in adults (fatty acid-binding protein, S100 A6, S100 A7, S100 A9, prolactin-inducible protein, Ig kappa chain, cystatin SN, cystatin S/SA and α-amylase 1). Four spots, already detected but not characterized by other authors in human saliva 2-DE, were attributed to different protein species of S100 A9 (long-type and long-type monophosphorylated, short-type and short-type monophosphorylated) by MS/MS analysis of tryptic peptides and sequential staining of 2-DE gels with Pro-Q Diamond, for specific detection of phosphoproteins, and total protein SYPRO Ruby stain.

Significance: Differential protein expression analysis of the acid insoluble fraction of saliva from preterm, at-term newborns and adults has been performed in this study by coupling 2-DE analysis and high-resolution tandem mass spectrometry in order to complete the information previously obtained by top-down LC–MS only on the acid-soluble proteome. Several proteins identified in the acid insoluble fraction of both preterm newborn and adult saliva are not of glandular origin, being only prolactin-inducible protein, salivary cystatins, α-amylase and polymeric immunoglobulin receptor exclusive of salivary glands. Three proteins resulted increased in at-term newborns with respect to preterm newborns and adults: BPI fold-containing family A member 1, two proteoforms of annexin A1 and keratin type 1 cytoskeletal 13, while several proteins were significantly increased in adults.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jprot.2016.06.021DOI Listing
September 2016