Search

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.

Background Goat cheese is an important element of the Mediterranean diet, appreciated for its health-promoting features and the typical gustatory essences. A pivotal role in the development of these characteristics is attributed to the hosted microbiota and its continuous remodeling over the space and time. Nevertheless, a thorough study of the cheese-associated microbiota by two meta-omics approaches is still missing. Also, the study of these model systems is important in the One Health context as they enable the development of systems for monitoring environmental micro-organisms and their biological variability. In this study we employed 16S rRNA gene sequencing and metaproteomics to explore the microbiota of a typical raw goat milk cheese at diverse ripening timepoints and depths of the cheese wheel. Results findings from 16S rRNA gene sequencing and metaproteomics described a stable microbiota ecology across the selected ripening timepoints, providing evidence on the microbiologically driven fermentation of the goat milk products. On the other hand, important modeling of the microbiota harbored in the surface and core of the cheese mass are highlighted both in compositional and functional terms. Conclusion observed outcomes portrait the rind microbiota struggling for the maintenance of the cheese biosafety through competition mechanisms and/or preventing the cheese colonization by pathobionts of animal or environmental origin. Efforts in microbial competition are also accomplished in the core microbiota, although its further focusing on other biochemical routes supports the role of this microbiota in the development of both the health beneficial effects and the pleasant gustatory nuances of the goat cheeses.

An enigmatic and never described hyper-reactivity of most of the cysteines resident in the reduced, molten globule-like intermediate of a few proteins has been recently discovered. In particular, all ten cysteines of chymotrypsinogen showed hundred times increased reactivity against hydrophobic reagents. A single cysteine (Cys1) was also found thousand times more reactive toward GSSG, making speculate that a single glutathionylation could represent the primordial event of its oxidative folding. In the present study, we compare these kinetic properties with those present in trypsinogen taken in its reduced, molten globule-like intermediate and identify the origin of these unusual properties. Despite the divergent evolution of these two proteins, the different amount of disulfides and the very different three-dimensional localization of three disulfides, their hyper-reactivity toward hydrophobic thiol reagents and disulfides is very similar. Mass spectrometry identifies two cysteines in trypsinogen, Cys148 and Cys197, 800 times more reactive toward GSSG than an unperturbed protein cysteine. These results point towards a stringent and accurate preservation of these peculiar kinetic properties during a divergent evolution suggesting some important role which at the present can only be hypothesized. Similar extraordinary hyper-reactivity has been found also in albumin, ribonuclease and lysozyme confirming that it cannot be considered a kinetic singularity of a single protein. Interestingly, the very flexible and fluctuating structures like those typical of the molten globule status proves capable of enabling sophisticated actions typical of enzymes like binding to GSSG with relevant specificity and high affinity (KD = 0.4 mM) and accelerating the reaction of its cysteines by thousands of times.

Alzheimer disease (AD) is the most prevalent neurodegenerative disease in the elderly, characterized by accumulation in the brain of misfolded proteins, inflammation, and oxidative damage leading to neuronal cell death. By considering the viewpoint that AD onset and worsening may be influenced by environmental factors causing infection, oxidative stress, and inflammatory reaction, we investigated the changes of the salivary proteome in a population of patients with respect to that in healthy controls (HCs). Indeed, the possible use of saliva as a diagnostic tool has been explored in several oral and systemic diseases. Moreover, the oral cavity continuously established adaptative and protective processes toward exogenous stimuli. In the present study, qualitative/quantitative variations of 56 salivary proteoforms, including post-translationally modified derivatives, have been analyzed by RP-HPLC-ESI-IT-MS and MS/MS analyses, and immunological methods were applied to validate MS results. The salivary protein profile of AD patients was characterized by significantly higher levels of some multifaceted proteins and peptides that were either specific to the oral cavity or also expressed in other body districts: (i) peptides involved in the homeostasis of the oral cavity; (ii) proteins acting as ROS/RNS scavengers and with a neuroprotective role, such as S100A8, S100A9, and their glutathionylated and nitrosylated proteoforms; cystatin B and glutathionylated and dimeric derivatives; (iii) proteins with antimicrobial activity, such as α-defensins, cystatins A and B, histatin 1, statherin, and thymosin β4, this last with a neuroprotective role at the level of microglia. These results suggested that, in response to injured conditions, Alzheimer patients established defensive mechanisms detectable at the oral level. Data are available via ProteomeXchange with identifier PXD021538.

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.

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.

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 dark proteins. 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.

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.

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.

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., Graphical abstract Image 1

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 Des1–4, cystatin SN P11 → L variant, and cystatin A T96 → 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.

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.

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.

Saliva contains hundreds of small proline-rich peptides most of which derive from the post-translational and post-secretory processing of the acidic and basic salivary praline-rich proteins. Among these peptides we found that a 20 residue proline-rich peptide (p1932), commonly present in human saliva and patented for its antiviral activity, was internalized within cells of the oral mucosa. The cell-penetrating properties of p1932 have been studied in a primary gingival fibroblast cell line and in a squamous cancer cell line, and compared to its retro-inverso form. We observed by mass-spectrometry, flow cytometry and confocal microscopy that both peptides were internalized in the two cell lines on a time scale of minutes, being the natural form more efficient than the retro-inverso one. The cytosolic localization was dependent on the cell type: both peptide forms were able to localize within nuclei of tumoral cells, but not in the nuclei of gingival fibroblasts. The uptake was shown to be dependent on the culture conditions used: peptide internalization was indeed effective in a complete medium than in a serum-free one allowing the hypothesis that the internalization could be dependent on the cell cycle. Both peptides were internalized likely by a lipid raft-mediated endocytosis mechanism as suggested by the reduced uptake in the presence of methyl-g-cyclodextrin. These results suggest that the natural peptide may play a role within the cells of the oral mucosa after its secretion and subsequent internalization. Furthermore, lack of cytotoxicity of both peptide forms highlights their possible application as novel drug delivery agents. (C) 2015 Elsevier B.V. All rights reserved.

Analysis by a HPLC-ESI-MS top-down proteornic platform of specimens of human preterm newborn whole saliva evidenced high relative amounts of cystatin B and its S-glutathionylated, S-cysteinylated, and S-S 2-mer (on Cys(3),) derivatives, decreasing as a function of postconceptional age (PCA). The percentage of S-unmodified cystatin B was higher than the S-modified isoforms in the early PCA period, differently from adults where cystatin B was detectable only as S-modified derivatives. The percentage of S-modified derivatives increased as a function of PCA, reaching at the normal term of delivery values similar to those determined in at-term newborns, babies, and adults. Moreover, in the early PCA period, high relative amounts of the 1-53 and 54-98 cystatin B fragments were detected, decreasing as a function of PCA and disappearing at the normal term of delivery. In agreement with intact cystatin B, fragment 1-53 was detectable as S-unmodified and S-modified derivatives, and their percentages changed accordingly with the percentages of intact proteins, suggesting that the fragmentation process could be subsequent to and independent from the S-modification of the protein. This study highlights specific enzymatic activity in the oral cavity of preterm newborns not present in at-term newborns and adults, which can be a clue to specialized pathways occurring during fetal oral development.

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 pKa 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.

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.

The purpose of our work was to identify unknown interaction partners of thymosin β4 (Tβ4). It was suggested that Tβ4 could be an antifibrotic drug for treatment of liver fibrogenesis, because Tβ4 prevents the platelet-derived growth factor-BB (PDGF-BB)-induced activation of hepatic stellate cells (HSCs). Very little information is available how Tβ4 counteracts the PDGF-BB-induced activation of HSCs. We propose the hypothesis that Tβ4 could bind directly to PDGF-BB and thereby reduce the concentration of free PDGF-BB available for binding to the PDGF-β receptor.To prove our suggestion of a direct interaction between Tβ4 and PDGF-BB, we carried out chemical as well as photochemical cross-linking experiments between the two pure proteins in vitro.We identified an interaction between Tβ4 and PDGF-BB by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) cross-linking as well as through biotin label transfer using a bifunctional photoactivatable derivative of Tβ4. In an in vitro system, PDGF-BB was identified as the first extracellular partner interacting with Tβ4. This interaction could influence PDGF-BB binding to its receptor and abolish PDGF-BB-related effects.Direct interaction of Tβ4 with extracellular factors should be considered as a potential mechanism to explain the pleiotropic effects of β-thymosins.

A top-down/bottom-up integrated proteomic approach based on LC-MS and 2-DE analysis was applied for comparative characterization of medulloblastoma and pilocytic astrocytoma posterior cranial fossa pediatric brain tumor tissues. Although rare, primary brain tumors are the most frequent solid tumors in the pediatric age. Among them the medulloblastoma is the prevalent malignant tumor in childhood while pilocytic astrocytoma is the most common, rarely showing a malignant progression. Due to the limited availability of this kind of sample, the study was applied to pooled tumor tissues for a preliminary investigation. The results showed different proteomic profiles of the two tumors and evidenced interesting differential expression of several proteins and peptides. Top-down proteomics of acid-soluble fractions of brain tumor homogenates ascribed a potential biomarker role of malignancy to beta- and alpha-thymosins and their truncated proteoforms and to C-terminal truncated (des-GG) ubiquitin, resulting exclusively detected or over-expressed in the highly malignant medulloblastoma. The bottom-up proteomics of the acid-soluble fraction identified several proteins, some of them in common with 2-DE analysis of acid-insoluble pellets. Peroxiredoxin-1, peptidyl-prolyl cis-trans isomerase A, triosephosphate isomerase, pyruvate kinase PKM, tubulin beta and alpha chains, heat shock protein HSP-90-beta and different histones characterized the medulloblastoma while the Ig kappa chain C region, serotransferrin, tubulin beta 2A chain and vimentin the pilocytic astrocytoma. The two proteomic strategies, with their pros and cons, well complemented each other in characterizing the proteome of brain tumor tissues and in disclosing potential disease biomarkers to be validated in a future study on individual samples of both tumor histotypes.

An important contribution to the variability of any proteome is given by the time dimension that should be carefully considered to define physiological modifications. To this purpose, whole saliva proteome was investigated in a wide age range. Whole saliva was collected from 17 preterm newborns with a postconceptional age at birth of 178-217 days. In these subjects sample collection was performed serially starting immediately after birth and within about 1 year follow-up, gathering a total of 111 specimens. Furthermore, whole saliva was collected from 182 subjects aged between 0 and 17 years and from 23 adults aged between 27 and 57 years. The naturally occurring intact salivary proteome of the 316 samples was analyzed by low- and high-resolution HPLC-ESI-MS platforms. Proteins peculiar of the adults appeared in saliva with different time courses during human development. Acidic proline-rich proteins encoded by PRH2 locus and glycosylated basic proline-rich proteins encoded by PRB3 locus appeared following 180 days of postconceptional age, followed at 7 months (+/- 2 weeks) by histatin 1, statherin, and P-B peptide. The other histatins and acidic proline-rich proteins encoded by PRH1 locus appeared in whole saliva of babies from 1 to 3 weeks after the normal term of delivery, S-type cystatins appeared at 1 year (+/- 3 months), and basic proline-rich proteins appeared at 4 years (+/- 1 year) of age. All of the proteinases involved in the maturation of salivary proteins were more active in preterm than in at-term newborns, on the basis of the truncated forms detected. The activity of the Fam20C kinase, involved in the phosphorylation of various proteins, started around 180 days of postconceptional age, slowly increased reaching values comparable to adults at about 2 years (+/- 6 months) of age. Instead, MAPK14 involved in the phosphorylation of S100A9 was fully active since birth also in preterm newborns.

Wilson's disease is a rare inherited disorder of copper metabolism, manifesting hepatic, neurological and psychiatric symptoms. Early diagnosis is often unfeasible and a unique diagnostic test is currently inapplicable. We performed the qualitative/quantitative characterization of the salivary proteome/peptidome of 32 Wilson's disease patients by an integrated top-down/bottom-up approach. Patients exhibited significant higher levels of S100A9 and S100A8 proteoforms, and their oxidized forms with respect to controls. Oxidation occurred on methionine and tryptophan residues, and on the unique cysteine residue, in position 42 in S100A8, and 3 in S100A9, that generated glutathionylated, cysteinylated, sulfinic, sulfonic, and disulfide dimeric forms. Wilson's disease patient saliva showed high levels of two new fragments of the polymeric immunoglobulin receptor, and of alpha-defensins 2 and 4. Overall, the salivary proteome of Wilson's disease patients reflected oxidative stress and inflammatory conditions characteristic of the pathology, highlighting differences that could be useful clues of disease exacerbation. (C) 2015 Elsevier B.V. All rights reserved.

The aim of this study was to characterize β and α thymosins and their proteoforms in various tissues and bodily fluids by mass spectrometry and to look at their association with a wide variety of pathologies.A top-down proteomic platform based on high-performance liquid chromatography (HPLC) coupled to high-resolution LTQ-Orbitrap mass spectrometry (MS) was applied to the characterization of naturally occurring peptides.In addition to thymosin β4 (Tβ4) and β10 (Tβ10), several post-translational modifications of both these peptides were identified not only in bodily fluids but also in normal and pathological tissues of different origins. The analysis of tissue specimens allowed the characterization of different C-terminal truncated forms of Tβ4 and Tβ10 together with other proteolytic fragments. The sulfoxide derivative of both Tβ4 and Tβ10 and the acetylated derivatives at lysine residues of Tβ4 were also characterized. Different proteoforms of prothymosin α, parathymosin α, thymosin α1 and thymosin α11 together with diverse proteolytic fragments were identified too.The clinical and prognostic significance and the origin of these proteoforms have to be deeply investigated.

We previously showed that the mutant strain of Enterococcus faecalis lacking the transcriptional regulator SlyA is more virulent than the parental strain. We hypothesized that this phenotype was due to overexpression of the second gene of the slyA operon, ef_3001 , renamed pmvE (for p olyamine m etabolism and v irulence of E. faecalis ). PmvE shares strong homologies with N 1 -spermidine/spermine acetyltransferase enzymes involved in the metabolism of polyamines. In this study, we used an E. faecalis strain carrying the recombinant plasmid pMSP3535- pmvE (V19/p3535- pmvE ), which allows the induction of pmvE by addition of nisin. Thereby, we showed that the overexpression of PmvE increased the virulence of E. faecalis in the Galleria mellonella infection model, as well as the persistence within peritoneal macrophages. We were also able to show a direct interaction between the His-tagged recombinant PmvE (rPmvE) protein and putrescine by the surface plasmon resonance (SPR) technique on a Biacore instrument. Moreover, biochemical assays showed that PmvE possesses an N -acetyltransferase activity toward polyamine substrates. Our results suggest that PmvE contributes to the virulence of E. faecalis , likely through its involvement in the polyamine metabolism.

The naturally occurring peptides, mainly arising from the proteolytic cleavage of larger proteins, play several functions within the body (e. g. antihypertensive, immuno-modulatory, anti-microbial and antiviral, mineral carriers). Their presence or the increase of their concentration could be connected to different pathologies and thereby some peptides could be useful biomarkers for the diagnosis or prognosis of the disease. Peptidome research, particularly within biological fluids, therefore represents one of the most interesting and challenging purposes of proteomics. In this review we describe the current state-of-the-art in peptidomics-based studies of several human bodily fluids (serum, plasma, urine, cerebrospinal fluid, saliva, tears, seminal fluid, vitreous humor, pancreatic juice), emphasizing the contribution of top-down proteomic platforms to the deep structural characterization of natural peptides and their posttranslational modifications.

Liquid chromatography in coupling with high-resolution ESI-LTQ-Orbitrap mass spectrometry was applied for a proteomic study of pediatric pilocytic astrocytoma brain tumor intracystic fluid by an integrated top-down/bottom-up platform. Both of the proteomic strategies resulted complementary and support each other in contributing to a wide characterization of the protein and peptide content of the tumor fluid. Top-down approach allowed to identify several proteins and peptides involved in different biological activities together with the characterization of interesting proteoforms such as fibrinopeptide A and its truncated form, fibrinopeptide B, complement C3f fragments, β-thymosin peptides, ubiquitin, several apolipoproteins belonging to A and C families, apolipoprotein J and D, and cystatin C. Of particular interest resulted the identification of a N-terminal truncated cystatin C proteoform, likely involved in immune response mechanism modulations and the identification of oxidized and glycosylated apolipoproteins including disulfide bridge dimeric forms. The bottom-up approach confirmed some of the experimental data findings together with adding the characterization of high-molecular-mass proteins in the samples. These data could contribute to elucidate the molecular mechanisms involved in onset and progression of the disease and cyst development.

Comprehensive analysis and characterization of the human salivary proteome is an important step towards the possible use of saliva for diagnostic and prognostic purposes. The contribution of the different sources to whole saliva, and the evaluation of individual variability and physiological modifications have been investigated by top-down proteomic approaches, disclosing the faceted and complex profile of the human salivary proteome. All this information is essential to develop saliva protein biomarkers. In this Review the major results obtained in the field by top-down platforms, and the improvements required to allow a more complete picture, will be discussed.

A new photoactivatable trifunctional cross-linker, cBED (cadaverine-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3'-dithiopropionate), was synthesized by chemical conversion of sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3'-dithiopropionate) with cadaverine. This cross-linker was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) and characterized using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. cBED is based on sulfo-SBED that has a photoactivatable azido group, a cleavable disulfide bond for label transfer methods, and a biotin moiety for highly sensitive biotin/avidin detection. By ultraviolet (UV) light, the azido group is converted to a reactive nitrene, transforming transient bindings of interacting structures to covalent bonds. In contrast to the sulfo-N-hydroxysuccinimide (sulfo-NHS) moiety of sulfo-SBED, which attaches quite unspecifically to amino groups, cBED includes a cadaverine moiety that can be attached by transglutaminase more specifically to certain glutamine residues. For instance, thymosin β4 can be labeled with cBED using tissue transglutaminase. By high-resolution HPLC/ESI-MS (electrospray ionization-mass spectrometry) and tandem MS (MS/MS) of the trypsin digest, it was established that glutamine residues at positions 23 and 36 were labeled, whereas Q39 showed no reactivity. The covalent binding of cBED to thymosin β4 did not influence its G-actin sequestering activity, and the complex could be used to identify new interaction partners. Therefore, cBED can be used to better understand the multifunctional role of thymosin β4 as well as of other proteins and peptides.

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 ?-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. © 2014 Elsevier B.V.

Glutathione transferases (GSTs) are a superfamily of detoxifying enzymes over-expressed in tumor tissues and tentatively proposed as biomarkers for localizing and monitoring injury of specific tissues. Only scarce and contradictory reports exist about the presence and the level of these enzymes in human saliva. This study shows that GSTP1-1 is the most abundant salivary GST isoenzyme, mainly coming from salivary glands. Surprisingly, its activity is completely obscured by the presence of a strong oxidizing agent in saliva that causes a fast and complete, but reversible, inactivation. Although salivary alpha-defensins are also able to inhibit the enzyme causing a peculiar half-site inactivation, a number of approaches (mass spectrometry, site directed mutagenesis, chromatographic and spectrophotometric data) indicated that hypothiocyanite is the main salivary inhibitor of GSTP1-1. Cys47 and Cys101, the most reactive sulfhydryls of GSTP1-1, are mainly involved in a redox interaction which leads to the formation of an intra-chain disulfide bridge. A reactivation procedure has been optimized and used to quantify GSTP1-1 in saliva of 30 healthy subjects with results of 42±4 mU/mg-protein. The present study represents a first indication that salivary GSTP1-1 may have a different and hitherto unknown function. In addition it fulfills the basis for future investigations finalized to check the salivary GSTP1-1 as a diagnostic biomarker for diseases.

Candida mannan (Mn) detection in bronchoalveolar lavage fluid (BALF) was shown to be useful for earlier identification and preemptive therapy targeting in preterm infants at high risk of invasive Candida infection. We investigated whether early detection of Candida Mn in BALF is associated with the presence of some neutrophilic products, as markers of prenatal infection/inflammation.BALF specimens were collected during the first 48 h of life from mechanically ventilated preterm newborns. Samples were analyzed by high-performance liquid chromatography-electrospray ionization-mass spectrometry. The relative amounts of α-defensins 1-4 and S100A proteins were measured by extracted ion current peak area. Absolute and differential white cell counts in BALF were obtained. Mn antigen concentrations were determined by the Platelia Candida antigen kit.Twenty-five studied neonates were divided into two groups: Mn-positive group and Mn-negative group. Levels of α-defensins 1-4 and S100A12 were significantly higher in the Mn-positive group than in the Mn-negative group. Moreover, positive significant correlations between the absolute number of neutrophils and the levels of α-defensins 1-4 and S100A8 were observed.The detection of Mn antigen in BALF of preterm infants is consistent with evidence of an innate immune response in their lungs as demonstrated by higher levels of α-defensins and S100A proteins.

People with Down syndrome, a frequent genetic disorder in humans, have increased risk of health problems associated with this condition. One clinical feature of Down syndrome is the increased prevalence and severity of periodontal disease in comparison with the general population. Because saliva plays an important role in maintaining oral health, in the present study the salivary proteome of Down syndrome subjects was investigated to explore modifications with respect to healthy subjects. Whole saliva of 36 Down syndrome subjects, divided in the age groups 10-17 yr and 18-50 yr, was analyzed by a top-down proteomic approach, based on the high performance liquid chromatography-electrospray ionization-MS analysis of the intact proteins and peptides, and the qualitative and quantitative profiles were compared with sex-and age-matched control groups. The results showed the following interesting features: 1) as opposed to controls, in Down syndrome subjects the concentration of the major salivary proteins of gland origin did not increase with age; as a consequence concentration of acidic proline rich proteins and S cystatins were found significantly reduced in older Down syndrome subjects with respect to matched controls; 2) levels of the antimicrobial alpha-defensins 1 and 2 and histatins 3 and 5 were significantly increased in whole saliva of older Down syndrome subjects with respect to controls; 3) S100A7, S100A8, and S100A12 levels were significantly increased in whole saliva of Down syndrome subjects in comparison with controls. The increased level of S100A7 and S100A12 may be of particular interest as a biomarker of early onset Alzheimer's disease, which is frequently associated with Down syndrome.

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 O2-affinity. In the presence of both chloride and organic phosphates, O2-affinity was strongly reduced, and cooperativity was enhanced; moreover, cathodic hemoglobin contains two indistinguishable GTP-binding sites. Stripped anodic hemoglobins exhibited both low O2-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.

Graft-versus-host disease (GVHD) is the major life-threatening complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT), developing in 35%-70% of all allo-HSCT recipients despite immunosuppressive prophylaxis. The recent application of proteomic tools that allow screening for differentially expressed or excreted proteins in body fluids could possibly identify specific biomarkers for GVHD. Whole saliva is highly attractive for noninvasive specimen collection. In the present study, we collected saliva specimens from 40 consecutives patients who underwent allo-HSCT between December 2008 and March 2011 at our institution. The specimens were analyzed by HPLC coupled to electrospray-ionization mass spectrometry. Variable expression of S100 protein family members (S100A8, S100A9, and S100A7) was detected. Fourteen of 23 patients with GVHD demonstrated the presence of S100A8, compared with only 2 patients without GVHD and 0 patients in the control group (P=.001). S100A7 was detectable in 11 of the 23 patients with GVHD but was absent in the other 2 groups (P=.0001). S100A9-short was detected in 20 patients with GVHD, in 9 patients without GVHD, and in 8 healthy volunteers (P=.01) Further studies are needed to clarify the role of these proteins as a marker of GVHD or as an index of mucosal inflammation. (C) 2013 American Society for Blood and Marrow Transplantation.

Dystroglycan (DG) is a membrane-associated protein complex formed by two noncovalently linked subunits, a-DG, a highly glycosylated extracellular protein, and beta-DG, a transmembrane protein. The interface between the two DG subunits, which is crucial to maintain the integrity of the plasma membrane, involves the C-terminal domain of a-DG and the N-terminal extracellular domain of beta-DG. It is well known that under both, physiological and pathological conditions, gelatinases (i.e. MMP-9 and/or MMP-2) can degrade DG, disrupting the connection between the extracellular matrix and the cytoskeleton. However, the molecular mechanisms and the exact cleavage sites underlying these events are still largely unknown. In a previous study, we have characterized the enzymatic digestion of the murine beta-DG ectodomain by gelatinases, identifying a main cleavage site on the beta-DG ectodomain produced by MMP-9. In this article, we have deepened the pattern of the beta-DG ectodomain digestion by MMP-2 by using a combined approach based on SDS-PAGE, Orbitrap, and HPLC-ESI-IT mass spectrometry. Furthermore, we have characterized the kineticparameters of the digestion of some beta-DG ectodomain mutants by gelatinases. (C) 2012 IUBMB IUBMB Life, 64(12): 988994, 2012

Background β-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.

Sjogren's syndrome (SS) is a chronic, inflammatory, autoimmune disease characterized by lymphocytic infiltration of the exocrine glands leading to qualitatively altered and diminished or absent salivary and lachrymal secretion, and by marked B-cell hyperreactivity. Many efforts have been made to define a panel of salivary and lachrymal markers helpful to design diagnostic tests able to replace blood tests and tissue biopsies for the diagnosis of primary and secondary SS. Several proteomic-based studies have indicated that a number of proteins and peptides can be considered SS biomarkers, being 2–3-fold up- or down-regulated compared to normal subject or having an exclusive presence in the saliva or tears of SS patients. Unfortunately, several factors make it difficult to define a comprehensive salivary and lachrymal panel of markers of SS, as the lack of a comprehensive proteomic analysis of human tears and saliva of healthy subjects, the lack of uniform protocols to collect and treat these samples, and the high grade of posttranslational modification of the proteins in these fluids.

Cigarette smoking plays an important role as a cause of morbidity and mortality in humans, involving respiratory, cardiovascular, digestive and reproductive systems. Tobacco smoke contains a large number of molecules, some of which are proven carcinogens. Although not fully understood, polymorphonuclear leukocytes seem to play a crucial role in the mechanisms by which tobacco smoke compounds are implicated in smoke-related diseases. In this paper the effects of an aqueous cigarette smoke extract on the expression of adhesion molecules of polymorphonuclear leukocytes together with the changes in the cell morphology have been related to the chemiluminescence activity. The results obtained show that polymorphonuclear leukocytes treated with aqueous cigarette smoke extract are significantly impaired, as suggested by the changes of chemiluminescence activity, of membrane receptors (CD18, CD62), myeloperoxidase expression and of cell morphology. Altogether the present data indicate that treated polymorphonuclear leukocytes are ineffectively activated and therefore unable to phagocytize zymosan particles. Copyright © 2010 John Wiley & Sons, Ltd.

In this review, the recent contribution of CE-MS technology to the analysis of amino acids, as well as the advantages of the hyphenation and the technologies involved in the instrumental coupling are reported. Different sections are dedicated to the recent contributions of CE-MS to the analysis of protein amino acids and their post-translational modifications, such as phosphorylation and sulfation. CE-MS analysis of some amino acid derivatives, such as the free methylated-derivatives of arginine is also discussed. A section is specifically devoted to the CE-MS applications in the field of chiral separation of D- and L-amino acid enantiomers.

Saliva is a body fluid of a unique composition devoted to protect the mouth cavity and the digestive tract. Our high performance liquid chromatography (HPLC)-electrospray ionization-MS analysis of the acidic soluble fraction of saliva from preterm human newborn surprisingly revealed more than 40 protein masses often undetected in adult saliva. We were able to identify the following proteins: stefin A and stefin B, S100A7 (two isoforms), S100A8, S100A9 (four isoforms), S100A11, S100A12, small proline-rich protein 3 (two isoforms), lysozyme C, thymosins beta(4) and beta(10), antileukoproteinase, histone H1c, and alpha and gamma globins. The average mass value reported in international data banks was often incongruent with our experimental results mostly because of post-translational modifications of the proteins, e.g. acetylation of the N-terminal residue. A quantitative label-free MS analysis showed protein levels altered in relation to the postconceptional age and suggested coordinate and hierarchical functions for these proteins during development. In summary, this study shows for the first time that analysis of these proteins in saliva of preterm newborns might represent a noninvasive way to obtain precious information of the molecular mechanisms of development of human fetal oral structures. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.003467, 1-14, 2011.