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A salivary proline-rich peptide of 1932 Da showed a dose-dependent antagonistic effect on the cytosolic Ca2+ mobilization induced by progesterone in a tongue squamous carcinoma cell line. Structure-activity studies showed that the activity of the peptide resides in the C-terminal region characterized by a proline stretch flanked by basic residues. Furthermore, lack of activity of the retro-inverso peptide analogue suggested the involvement of stereospecific recognition. Mass spectrometry-based shotgun analysis, combined with Western blotting tests and biochemical data obtained with the Progesterone Receptor Membrane Component 1 (PGRMC1) inhibitor AG205, showed strong evidence that p1932 performs its modulatory action through an interaction with the progesterone receptor PGRMC1, which is predominantly expressed in this cell line and, clearly, plays a role in progesterone induced Ca2+ response. Thus, our results point to p1932 as a modulator of the transduction signal pathway mediated by this protein and, given a well-established involvement of PGRMC1 in tumorigenesis, highlight a possible therapeutic potential of p1932 for the treatment of oral cancer. [ABSTRACT FROM AUTHOR]

During a survey of human saliva by a top-down reversed-phase high-performance liquid chromatography with electrospray ionization mass spectrometry approach, two proteins eluting at 27.4 and 28.4 min, with average masses of 15 494 ± 1 and 11 142 ± 1 Da, were detected in a subject from Boston. The Δmass value (4352 Da) of the two proteins was similar to the difference in mass values between intact (150 amino acids, [a.a.]) and truncated acidic proline-rich proteins (a PRPs; 106 a.a.) suggesting an a.a. substitution in the first 106 residues resulting in a strong reduction in polarity, since under the same experimental conditions a PRPs eluted at ∼22.5 min (intact) and 23.5 min (truncated forms). Manual inspection of the high-resolution high-performance liquid chromatography with electrospray ionization tandem mass spectra of the truncated isoform showed the replacement of the phosphorylated Ser-22 in PRP-3 with a Phe residue. Inspection of the tandem mass spectra of the intact isoform confirmed the substitution, which is allowed by the code transition TCT→ TTT and is in agreement with the dramatic increase in elution time. The isoform was also detected in two other subjects, one from Boston (unrelated to the previous) and one from Rome. For this reason we propose to name this variant PRP-1 ( PRP-3) RB (Roma-Boston) Ser22(phos)→Phe. [ABSTRACT FROM AUTHOR]

Abstract: The analysis of whole saliva of 32 subjects with diagnosis of schizophrenia (SZ), 17 with diagnosis of bipolar disorder (BD), and 31 healthy subjects divided in non-smokers (HN; n=19) and smokers (HS; n=12) using an HPLC–ESI-MS top-down platform is reported in this study. Both SZ and BD revealed more than 10 fold mean increase of α-defensins 1–4, S100A12, cystatin A and S-derivatives of cystatin B levels with respect to the HN and HS control groups. No differences of protein levels were observed between SZ and BD groups and between HN and HS groups. Moreover, the correlation coefficients among the different proteins were significantly better in BD group than in SZ group. Biological significance: This study on whole saliva confirms a schizophrenia-associated dysregulation of immune pathway of peripheral white blood cells and suggests that the dysregulation of BD group could involve the activation of more specific cell type than that of SZ group. [Copyright &y& Elsevier]

The hemoglobin system of the serpent eel Ophisurus serpens was structurally and functionally characterized with the aim of comparing it to the hemoglobin system of other fish species, as oxygen loading under the severe habitat conditions experienced by O. serpens could have necessitated specific adaptation mechanisms during evolution. The hemoglobin system of O. serpens includes one cathodic and four anodic components. The molecular mass of the α and β chains of the cathodic component as well as the 2 α and 4 β of the anodic components were determined. Analysis of the intact α and β chains from cathodic hemoglobin and their proteolytic digestion products by high-resolution MS and MS/MS experiments resulted in 92 and 95 % sequence coverage of the α and β globins, respectively. The oxygen binding properties of both hemoglobin components were analyzed with respect to their interactions with their physiological effectors. Stripped cathodic hemoglobin displayed the highest oxygen affinity among Anguilliformes with no significant effect of pH on O-affinity. In the presence of both chloride and organic phosphates, O-affinity was strongly reduced, and cooperativity was enhanced; moreover, cathodic hemoglobin contains two indistinguishable GTP-binding sites. Stripped anodic hemoglobins exhibited both low O-affinity and low cooperativity and a larger Bohr effect than cathodic hemoglobin. The cathodic hemoglobin of O. serpens and the corresponding component of Conger conger share the greatest structural and functional similarity among hemoglobin systems of Anguilliformes studied to date, consistent with their phylogenetic relationship. [ABSTRACT FROM AUTHOR]

Background: Adamantinomatous craniopharyngioma is the third most recurrent paediatric brain tumour. Although histologically benign, it behaves aggressively as a malignant tumour due to invasion of the hypothalamus and visual pathways. Surgery is still the first and almost the only mode of treatment, although serious damage can occur as a consequence of tumour localization. The proteomic characterization of the intracystic tumoural fluid could contribute to the comprehension of the tumorigenesis processes and to the development of therapeutic targets to reduce cyst volume, allowing less invasive surgery and/or delay of the radical resection of the tumour mass and the collateral serious effects. Methods: Intracystic fluid was analysed by a LC-ESI-IT-MS top-down platform after acidification, deproteinization and chloroform liquid/liquid extraction. Findings: Thymosin β and β peptides were for the first time identified in the intracystic fluid of adamantinomatous craniopharyngioma by low- and high-resolution MS analysis coupled with LC. The two peptides showed the same distribution trend in the analysed samples. Thymosin β and β were present in 77 % of the analysed samples. These peptides were not found in the cerebrospinal fluid available for two patients. Interpretation: The presence of β-thymosins in the intracystic fluid of the tumour confirmed the secretion of these proteins in the extracellular environment. Due to their G-actin-sequestering activity and antiapoptotic and anti-inflammatory properties, these peptides could be strictly involved in both tumour progression and cyst development and growth. [ABSTRACT FROM AUTHOR]

This review is addressed to scientists working outside the field of proteomics and wishes to shed a light on the possibility offered by the latest proteomics strategies. Bottom-up and top-down platforms are critically examined outlining advantages and limitations of their application to qualitative and quantitative investigations. Discovery, directed and targeted proteomics as different options for the management of the MS instrument are defined emphasizing their integration in the experimental plan to accomplish meaningful results. The issue of data validation is analyzed and discussed. The most common qualitative proteomic platforms are described, with a particular emphasis on enrichment methods to elucidate PTMs codes (i.e. ubiquitin and histone codes). Label-free and labeled methods for relative and absolute quantification are critically compared. The possible contribution of proteomics platforms to the transition from structural proteomics to functional proteomics (study of the functional connections between different proteins) and to the challenging system biology (integrated study of all the functional cellular functions) is also briefly discussed. [ABSTRACT FROM AUTHOR]

This study describes the characterization of the glycan moieties and the peptide backbone of six glycoforms of IB-8a CON1+, a basic proline-rich protein present in human saliva. MS analyses on the intact glycoproteins before and after N-deglycosylation with PNGase F and high-resolution MS/ MS sequencing by LTQ Orbitrap XL of peptides and glycopeptides from tryptic digests allowed the structural characterization of the glycan moieties and the polypeptide backbone, as well as to establish the glycosylation site at the asparagine residue at 98th position. Five of the glycoforms carry a biantennary N-linked glycan fucosylated in the innermost N-acetylglucosamine of the core and showing from zero to four additional fucoses in the antennal region. The sixth glycoform carries a monoantennary monofucosylated oligosaccharide. The glycoform cluster was detected on 28 of 71 adult saliva specimens. Level of fucosylation showed interindividual variability with the major relative abundance for the trifucosylated glycoform. Nonglycosylated IB-8a CON1+ and the variant IB-8a CON1−, lacking of the glycosylation site, have been also detected in human saliva. [ABSTRACT FROM AUTHOR]

Abstract: Stenotrophomonas maltophilia is a microorganism of environmental and clinical importance as well as a frequent airway colonizer of cystic fibrosis (CF) individuals. We combined 2-DE and MALDI-TOF MS to profile the protein expression in S. maltophilia K279a, a completely sequenced clinical isolate, grown at 37°C with respect to the strain grown at 26°C. Among the proteins up-regulated at 37°C, we identified GroEL, a molecular chaperone that mainly assist the folding and unfolding of proteins under both normal and stress conditions. A 2.4-kb groESL mRNA was detected independently by Northern blot analyses with a groES- and a groEL-specific probe, indicating that S. maltophilia groES and groEL form an operon. Primer extension analysis of S. maltophilia groESL done in Escherichia coli showed that 2 promoters, Pσ32 and Pσ70, were utilized under the heat-shock and normal condition, respectively, whereas S. maltophilia groEL was shown to act as a heat-shock gene at 37°C, 42°C, and, to a lesser extent, at 50°C by real-time RT-PCR analyses. Finally, immunoblot analyses revealed that S. maltophilia GroEL strongly reacted with sera from CF patients chronically infected by the microorganism, but did not with sera from CF patients with sporadic infection or uninfected. [Copyright &y& Elsevier]

Background: b-thymosins play roles in cytoskeleton rearrangement, angiogenesis, fibrosis and reparative process, thus suggesting a possible involvement in the pathogenesis of systemic sclerosis. The aim of the study was to investigate the presence of thymosins β4, β4 sulfoxide, and β10 in bronchoalveolar lavage fluid of scleroderma patients with interstitial lung disease and the relation of these factors with pulmonary functional and radiological parameters. Methods: β-thymosins concentrations were determined by Reverse Phase-High Performance Liquid Chromatography-Electrospray-Mass Spectrometry in the bronchoalveolar lavage fluid of 46 scleroderma patients with lung involvement and of 15 controls. Results: Thymosin β4, β4 sulfoxide, and β10 were detectable in bronchoalveolar lavage fluid of patients and controls. Thymosin β4 levels were significantly higher in scleroderma patients than in controls. In addition, analyzing the progression of scleroderma lung disease at one-year follow-up, we have found that higher thymosin β4 levels seem to have a protective role against lung tissue damage. Thymosin β4 sulfoxide levels were higher in the smokers and in the scleroderma patients with alveolitis. Conclusions: We describe for the first time β-thymosins in bronchoalveolar lavage fluid and their possible involvement in the pathogenesis of scleroderma lung disease. Thymosin β4 seems to have a protective role against lung tissue damage, while its oxidation product mirrors an alveolar inflammatory status. [ABSTRACT FROM AUTHOR]

Background: The best index of adrenal dysfunction is urinary free cortisol (UFC) measurements performed using a 24-h urine collection. This measurement is also useful in the investigation of Cushing's syndrome. In this paper, we report a simple and selective method for the analysis of UFC by liquid chromatography-tandem mass spectrometry (LC-MS/MS) suitable for use in a high-volume clinical laboratory routine. The results were compared to those obtained using a commercial immunoassay method used in our laboratory. Methods: Urine samples containing 50 ng of internal standard (Cortisol-9,11,12,12-d4) were deproteinized using centrifugal filters with a molecular weight 10,000 Da cut-off and injected on a reversed phase column. Cortisol was analyzed in highly selective reaction monitoring in positive atmospheric pressure chemical ionization mode, at a resolution of 0.4 amu full width half maximum, and following the transitions related to the precursor 363.2 for cortisol and 367.2 for deuterated cortisol. The method validation included analysis of precision, linearity, sensitivity, recovery and interference from structurally similar steroids. UFC from 230 subjects was measured using LC-MS/MS and electrochemiluminescence immunoassay (ECLIA) methods. Results: The calibration curves exhibited linearity and reproducibility in the range 7-10,000 nmol/L. Total imprecision was lower than 10%. The limit of detection and limit of quantification were 2 and 7 nmol/L, respectively. Mean recovery was higher than 90%. Structurally similar steroids do not interfere in the proposed method, but cause a significant change in the ECLIA results. Cortisol values obtained using the ECLIA method were always higher than those obtained using the LC-MS/MS method, with the bias directly proportional to cortisol concentrations. The reference values calculated using 180 normal subjects were 11-70 μg/day. Conclusions: The proposed method is sensitive, simple, free from interferences and reliable for routine use. Clin Chem Lab Med 2010;48:1433-7. [ABSTRACT FROM AUTHOR]

Goat cheese is an important element of the Mediterranean diet, appreciated for its health-promoting features and unique taste. A pivotal role in the development of these characteristics is attributed to the microbiota and its continuous remodeling over space and time. Nevertheless, no thorough study of the cheese-associated microbiota using two metaomics approaches has previously been conducted. Here, we employed 16S rRNA gene sequencing and metaproteomics to explore the microbiota of a typical raw goat milk cheese at various ripening timepoints and depths of the cheese wheel. The 16S rRNA gene-sequencing and metaproteomics results described a stable microbiota ecology across the selected ripening timepoints, providing evidence for the microbiologically driven fermentation of goat milk products. The important features of the microbiota harbored on the surface and in the core of the cheese mass were highlighted in both compositional and functional terms. We observed the rind microbiota struggling to maintain the biosafety of the cheese through competition mechanisms and/or by preventing the colonization of the cheese by pathobionts of animal or environmental origin. The core microbiota was focused on other biochemical processes, supporting its role in the development of both the health benefits and the pleasant gustatory nuances of goat cheese. [ABSTRACT FROM AUTHOR]

Basic and preclinical research founded the progress of personalized medicine by providing a prodigious amount of integrated profiling data and by enabling the development of biomedical applications to be implemented in patient-centered care and cures. If the rapid development of genomics research boosted the birth of personalized medicine, further development in omics technologies has more recently improved our understanding of the functional genome and its relevance in profiling patients' phenotypes and disorders. Concurrently, the rapid biotechnological advancement in diverse research areas enabled uncovering disease mechanisms and prompted the design of innovative biological treatments tailored to individual patient genotypes and phenotypes. Research in stem cells enabled clarifying their role in tissue degeneration and disease pathogenesis while providing novel tools toward the development of personalized regenerative medicine strategies. Meanwhile, the evolving field of integrated omics technologies ensured translating structural genomics information into actionable knowledge to trace detailed patients' molecular signatures. Finally, neuroscience research provided invaluable models to identify preclinical stages of brain diseases. This review aims at discussing relevant milestones in the scientific progress of basic and preclinical research areas that have considerably contributed to the personalized medicine revolution by bridging the bench-to-bed gap, focusing on stem cells, omics technologies, and neuroscience fields as paradigms. [ABSTRACT FROM AUTHOR]

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. Image 1 • Natural occurring proteolysis investigation of human salivary proteins by a top-down proteomics. • The study compared proteolytic events in pre-term, at-term newborns, babies and adults. • Maximum level of proteolysis preterm newborns by convertases and carboxypeptidases. [ABSTRACT FROM AUTHOR]

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. 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. • GCF was collected from deciduous, exfoliating and permanent teeth from 20 children. • Peptide/proteins content was characterized by a Top-Down MS proteomic approach. • The data help to understand complex biological processes like teeth eruption. • Data are available via ProteomeXcharge with identifier PXD016010 and PXD016049. Unlabelled Image [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

This review summarizes the results of a series of studies performed by our group with the aim to define the expression levels of thymosin β4 and thymosin β10 over time, starting from fetal development to different ages after birth, in different human organs and tissues. The first section describes the proteomics investigations performed on whole saliva from preterm newborns and gingival crevicular fluid, which revealed to us the importance of these acidic peptides and their multiple functions. These findings inspired us to start an in-depth investigation mainly based on immunochemistry to establish the distribution of thymosin β4 and thymosin β10 in different organs from adults and fetuses at different ages (after autopsy), and therefore to obtain suggestions on the functions of β-thymosins in health and disease. The functions of β-thymosins emerging from these studies, for instance, those performed during carcinogenesis, add significant details that could help to resolve the nowadays so-called "β-thymosin enigma", i.e., the potential molecular role played by these two pleiotropic peptides during human development. [ABSTRACT FROM AUTHOR]

Mitochondrial diseases (MDs) affect 4300 individuals, with different ages of presentation and manifestation in any organ. How defects in mitochondria can cause such a diverse range of human diseases remains poorly understood. In recent years, several published research articles regarding the metabolic and protein profiles of these neurogenetic disorders have helped shed light on the pathogenetic mechanisms. By investigating different pathways in MDs, often with the aim of identifying disease biomarkers, it is possible to identify molecular processes underlying the disease. In this perspective, omics technologies such as proteomics and metabolomics considered in this review, can support unresolved mitochondrial questions, helping to improve outcomes for patients. [ABSTRACT FROM AUTHOR]

Seminoma is the most common testicular cancer. Pituitary tumor-transforming gene 1 (PTTG1) is a securin showing oncogenic activity in several tumors. We previously demonstrated that nuclear PTTG1 promotes seminoma tumor invasion through its transcriptional activity on matrix metalloproteinase 2 (MMP-2) and E-cadherin (CDH1). We wondered if specific interactors could affect its subcellular distribution. To this aim, we investigated the PTTG1 interactome in seminoma cell lines showing different PTTG1 nuclear levels correlated with invasive properties. A proteomic approach upon PTTG1 immunoprecipitation uncovered new specific securin interactors. Western blot, confocal microscopy, cytoplasmic/nuclear fractionation, sphere-forming assay, and Atlas database interrogation were performed to validate the proteomic results and to investigate the interplay between PTTG1 and newly uncovered partners. We observed that spectrin beta-chain (SPTBN1) and PTTG1 were cofactors, with SPTBN1 anchoring the securin in the cytoplasm. SPTBN1 downregulation determined PTTG1 nuclear translocation, promoting its invasive capability. Moreover, a PTTG1 deletion mutant lacking SPTBN1 binding was strongly localized in the nucleus. The Atlas database revealed that seminomas that contained higher nuclear PTTG1 levels showed significantly lower SPTBN1 levels in comparison to non-seminomas. In human seminoma specimens, we found a strong PTTG1/SPTBN1 colocalization that decreases in areas with nuclear PTTG1 distribution. Overall, these results suggest that SPTBN1, along with PTTG1, is a potential prognostic factor useful in the clinical management of seminoma. [ABSTRACT FROM AUTHOR]