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1.
J Proteomics ; 226: 103890, 2020 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-32629195

RESUMO

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 ß4 and Thymosin ß10, 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 ß4 and Thymosin ß10; Fibrinopeptide A and its fragments and Fibrinopeptide B; S100A8 and S100A9, LVV Hemorphin-7 (hemoglobin chain ß fragment), as well as some other peptides deriving from α and ß 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.

2.
J Proteome Res ; 2020 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-32575983

RESUMO

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 ß4 and α-defensins 1 and 4 in SM-C patients with respect to SM+C. Data are available via ProteomeXchange with identifier PXD017759.

3.
J Clin Immunol ; 40(6): 840-850, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32519288

RESUMO

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.

4.
J Proteomics ; 222: 103797, 2020 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-32360370

RESUMO

The oral mucosal pellicle is a thin lubricating layer generated by the binding of saliva proteins on epithelial oral cells. The protein composition of this biological structure has been to date studied by targeted analyses of specific salivary proteins. In order to perform a more exhaustive proteome characterization of pellicles, we used TR146 cells expressing or not the transmembrane mucin MUC1 and generated pellicles by incubation with human saliva and washing to remove unbound proteins. A suitable method was established for the in vitro isolation of the mucosal pellicle by "shaving" it from the cells using trypsin. The extracts, the washing solutions and the saliva used to constitute the pellicles were analyzed by LC MS/MS (data are available via ProteomeXchange with identifier PXD017268). Comparison of pellicle and saliva compositions evidenced the adsorption of proteins not previously reported as pellicle constituents such as proteins of the PLUNC family. Pellicles formed on TR146 and TR146/MUC1 were also analyzed and compared by protein label-free quantification. The two types of samples appeared as distinct clusters in multivariate analyses, but the discriminant proteins (Welch test p < .05, FDR < 0.1) were cellular rather than salivary proteins. SIGNIFICANCE: The oral mucosal pellicle is made of salivary proteins tightly bound to oral epithelial cells. It is essential to oral health, with biological functions depending largely on its protein constituents. Characterizing its proteome is difficult due to the intimate association of this protein layer to cell membranes. In this work, we report a trypsin "shaving" protocol which enabled to sample the pellicle formed on an in vitro cellular model of oral epithelium. Analyzing such samples by high-resolution mass spectrometry provided novel information on the mucosal pellicle composition. This work is therefore a good starting point for further characterization of this biological structure.

5.
J Clin Immunol ; 40(2): 329-339, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31916122

RESUMO

PURPOSE: Present study is designed to discover potential salivary biomarkers associated with predominantly antibody deficiencies, which include a large spectrum of disorders sharing failure of antibody production, and B cell defects resulting in recurrent infections, autoimmune and inflammatory manifestations, and tumor susceptibility. Understanding and clinical classification of these syndromes is still challenging. METHODS: We carried out a study of human saliva based on liquid chromatography-mass spectrometry measurements of intact protein mass values. Salivary protein profiles of patients (n = 23) and healthy controls (n = 30) were compared. RESULTS: Patients exhibited lower abundance of α-defensins 1-4, cystatins S1 and S2, and higher abundance of glutathionylated cystatin B and cystatin SN than controls. Patients could be clustered in two groups on the basis of different levels of cystatin SN, S1 and S2, suggesting that these proteins may play different roles in the disease. CONCLUSIONS: Quantitative variations of these pro-inflammatory and antimicrobial peptides/proteins may be related to immunodeficiency and infectious condition of the patients. The high incidence of tumors in the group with the highest level of cystatin SN, which is recognized as tumoral marker, appeared an intriguing result deserving of future investigations. Data are available via ProteomeXchange with identifier PXD012688.

6.
J Proteome Res ; 19(1): 300-313, 2020 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-31638822

RESUMO

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 (P32 and A32 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: Q29 of P-H, Q37 of P-D, Q21 of II-2, Q41 of P-C, and Q37 of statherin were the principal reactive residues. One or two secondary glutamine residues of only P-H, P-D P32, 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.

7.
J Sep Sci ; 43(1): 313-336, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31631532

RESUMO

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.

8.
Arch Oral Biol ; 98: 148-155, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30496935

RESUMO

OBJECTIVE: In the present study the salivary proteome of burning mouth syndrome patients and healthy subjects was characterized by a top-down proteomic approach and compared to highlight possible qualitative and quantitative differences that may give suggestions about the causes of this pathology which are still unknown. MATERIALS AND METHODS: Resting and stimulated whole saliva, stimulated parotid and submandibular/sublingual saliva samples were collected from burning mouth syndrome patients (n = 16) and age- and gender-matched healthy subjects (n = 14). An equal volume of 0.2% trifluoroacetic acid was added to each sample immediately after collection and the supernatants were analysed by liquid chromatography coupled to electrospray-ionisation mass spectrometry. Proteins and peptides were quantified using a label-free approach measuring the extracted ion current peak areas of the main salivary proteins and peptides. RESULTS: The quantitation of the main salivary proteins and peptides revealed a higher concentration of cystatin SN in resting saliva of burning mouth syndrome patients with respect to healthy controls and no other conspicuous changes. CONCLUSIONS: The reported data showed that the salivary protein profile was not affected, in composition and relative abundance, by the burning mouth syndrome, except for the cystatin SN, a protein up-regulated in several pathological conditions, that might be considered potentially indicative of the disease.


Assuntos
Síndrome da Ardência Bucal/complicações , Síndrome da Ardência Bucal/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Saliva/química , Glândulas Salivares/química , Proteínas e Peptídeos Salivares/análise , Idoso , Cromatografia Líquida , Feminino , Humanos , Pessoa de Meia-Idade , Glândula Parótida/metabolismo , Salivação , Espectrometria de Massas por Ionização por Electrospray , Xerostomia/complicações
9.
J Proteomics ; 187: 212-222, 2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-30086402

RESUMO

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.


Assuntos
Esclerose Múltipla/metabolismo , Proteoma/análise , Proteômica/métodos , Proteínas e Peptídeos Salivares/análise , Adulto , Estudos de Casos e Controles , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Masculino , Espectrometria de Massas/métodos , Pessoa de Meia-Idade , Proteoma/metabolismo , Saliva/química , Saliva/metabolismo , Proteínas e Peptídeos Salivares/metabolismo
10.
J Proteome Res ; 17(9): 3292-3307, 2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30064219

RESUMO

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 S1 → A, P-Ko P36 → S, and P-Ko A41 → 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.


Assuntos
Processamento de Proteína Pós-Traducional , Saliva/química , Proteínas Salivares Ricas em Prolina/metabolismo , Adulto , Sequência de Aminoácidos , Cromatografia Líquida , Feminino , Glicosilação , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Glândula Parótida/química , Glândula Parótida/metabolismo , Peptídeos/análise , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Proteólise , Proteômica/métodos , Proteínas Salivares Ricas em Prolina/química , Proteínas Salivares Ricas em Prolina/genética , Proteínas Salivares Ricas em Prolina/isolamento & purificação , Espectrometria de Massas em Tandem
11.
Crit Rev Biochem Mol Biol ; 53(3): 246-263, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29564928

RESUMO

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.


Assuntos
Peptídeos , Proteínas de Plantas , Plantas , Animais , Humanos , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/química , Plantas/genética , Plantas/metabolismo
12.
J Proteome Res ; 17(1): 455-469, 2018 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-29083190

RESUMO

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.


Assuntos
Consumo de Bebidas Alcoólicas/genética , Proteoma/análise , Proteômica/métodos , Saliva/química , Animais , Itália , Ratos , Glândula Submandibular
13.
J Proteome Res ; 16(11): 4196-4207, 2017 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-29019242

RESUMO

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.


Assuntos
Polimorfismo Genético , Processamento de Proteína Pós-Traducional , Cistatinas Salivares/análise , Sequência de Aminoácidos , Inibidores de Cisteína Proteinase , Humanos , Espectrometria de Massas , Fosforilação , Saliva/química , Cistatinas Salivares/metabolismo
14.
Arch Oral Biol ; 77: 68-74, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28178587

RESUMO

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.


Assuntos
Proteínas Ricas em Prolina do Estrato Córneo/metabolismo , Cistatina A/metabolismo , Cistatina B/metabolismo , Proteômica/métodos , Saliva/química , Idoso , Estudos de Casos e Controles , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Boca Edêntula , Espectrometria de Massas por Ionização por Electrospray
15.
Physiol Behav ; 173: 163-173, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28130087

RESUMO

Individual differences in astringency perception are poorly understood. Astringency from tannins stimulates the release of specific classes of salivary proteins. These proteins form complexes with tannins, altering their perceived astringency and reducing their bioavailability. We studied the bitter compound, 6-n-propylthioural (PROP), as a phenotypic marker for variation in astringency perception and salivary protein responses. Seventy-nine subjects classified by PROP taster status rated cranberry juice cocktail (CJC; with added sugar) supplemented with 0, 1.5 or 2.0g/L tannic acid (TA). Saliva for protein analyses was collected at rest, or after stimulation with TA or cranberry juice (CJ; without added sugar). CJC with 1.5g/L tannic acid was found to be less astringent, and was liked more by PROP non-taster males than PROP taster males, consistent with the expectation that non-tasters are less sensitive to astringency. Levels of acidic Proline Rich Proteins (aPRPs) and basic Proline Rich Proteins (bPRPs) decreased after TA, while levels of aPRPs, bPRPs and Cystatins unexpectedly rose after CJ. Increases in bPRPs and Cystatins were only observed in PROP tasters. The PROP phenotype plays a gender-specific, but somewhat limited role in the perceived astringency of tannic-acid supplemented, cranberry juice cocktail. The PROP phenotype (regardless of gender) may also be involved in the release of salivary proteins previously implicated in oral health.


Assuntos
Adstringentes/farmacologia , Proteínas e Peptídeos Salivares/metabolismo , Percepção Gustatória/efeitos dos fármacos , Percepção Gustatória/fisiologia , Paladar/fisiologia , Uracila/análogos & derivados , Adolescente , Adulto , Análise de Variância , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Feminino , Sucos de Frutas e Vegetais , Humanos , Masculino , Espectrometria de Massas , Reconhecimento Psicológico , Caracteres Sexuais , Taninos/farmacologia , Adulto Jovem
16.
Expert Rev Proteomics ; 13(9): 883-94, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27498711

RESUMO

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.


Assuntos
Biomarcadores , Pediatria/tendências , Proteoma/genética , Proteômica , Adulto , Criança , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Metabolômica , Prognóstico
17.
J Proteomics ; 146: 48-57, 2016 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-27321913

RESUMO

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 HPLC­ESI­MS/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 LC­MS 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.


Assuntos
Recém-Nascido Prematuro/metabolismo , Proteoma/metabolismo , Saliva/metabolismo , Adolescente , Adulto , Anexina A1/análise , Eletroforese em Gel Bidimensional , Glicoproteínas/análise , Humanos , Lactente , Recém-Nascido , Queratina-1/análise , Pessoa de Meia-Idade , Fosfoproteínas/análise , Proteoma/análise , Saliva/química , Espectrometria de Massas em Tandem , Adulto Jovem
18.
J Sep Sci ; 39(10): 1987-97, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26991339

RESUMO

In the present study, we show that the heterogeneous mixture of glycoforms of the basic salivary proline-rich protein 3M, encoded by PRB3-M locus, is a major component of the acidic soluble fraction of human whole saliva in the first years of life. Reversed-phase high-performance liquid chromatography with high-resolution electrospray ionization mass spectrometry analysis of the intact proteoforms before and after N-deglycosylation with Peptide-N-Glycosidase F and tandem mass spectrometry sequencing of peptides obtained after Endoproteinase GluC digestion allowed the structural characterization of the peptide backbone and identification of N- and O-glycosylation sites. The heterogeneous mixture of the proteoforms derives from the combination of 8 different neutral and sialylated glycans O-linked to Threonine 50, and 33 different glycans N-linked to Asparagine residues at positions 66, 87, 108, 129, 150, 171, 192, and 213.


Assuntos
Polissacarídeos/química , Proteínas Salivares Ricas em Prolina/análise , Cromatografia Líquida de Alta Pressão , Glicosilação , Humanos , Espectrometria de Massas por Ionização por Electrospray
19.
PLoS One ; 11(1): e0147925, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26814504

RESUMO

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.


Assuntos
Cálcio/metabolismo , Peptídeos/metabolismo , Progesterona/farmacologia , Glândulas Salivares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sequência de Aminoácidos , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Citosol/metabolismo , Humanos , Íons/química , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Peptídeos/síntese química , Peptídeos/química , Progestinas/farmacologia , Domínios Proteicos Ricos em Prolina , Ligação Proteica , Receptores de Progesterona/antagonistas & inibidores , Receptores de Progesterona/metabolismo , Espectrometria de Massas por Ionização por Electrospray
20.
J Proteomics ; 134: 47-56, 2016 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-26375204

RESUMO

UNLABELLED: The most heterogeneous family of human salivary proteins is represented by proline-rich proteins (PRPs) divided in acidic, basic, and basic glycosylated (aPRPs, bPRPs, gPRPs). They are encoded by six genes, clustered on chromosome 12p13.2: PRH1-2 encode aPRPs, PRB1-4 encode bPRPs and gPRPs. Each gene exists in different allelic forms: two for PRH2, three for PRH1, PRB2, and PRB4, four for PRB1, and PRB3. During granule maturation, PRP proproteins undergo proteolysis by the action of convertases and carboxypeptidases. Differently from bPRPs, proteolysis of aPRPs is not complete, and, besides fragments, entire protein species are also secreted. Maturation process generates ten aPRPs (PRP-1, PRP-2, PIF-s, Db-s, Pa, PRP-3, PRP-4, PIF-f, Db-f, P-C), and at least 18 bPRPs (II-2, P-E, IB-6, Ps-1, Ps-2, IB-1, P-J, IB-8a, P-F, P-H, P-D, II-1, protein glycosylated A, CD-IIg, and Gl1-4). In addition, single nucleotide and length polymorphisms, and differentially spliced transcripts originate several natural variants. Phosphorylation, N-pyroglutaminylation, dimerization, and N-/O-glycosylation also occur during maturation, enlarging the number of protein species, further increased by proteolytic events governed by carboxy- and endo-peptidases during and after secretion, and giving rise to a huge number of small peptides. The PRP functional role is still poorly understood. SIGNIFICANCE: The high polymorphism of PRPs gives an important contribution to the high heterogeneity and inter-individual variability of the human salivary proteome. The products of six genes clustered on chromosome 12p13.2 comprise a mixture of entire, truncated, phosphorylated, glycosylated and dimerized protein/peptide species, sharing large part of their sequences, and possibly involved in different biological activities. Whatever the role of PRP species is, it should be crucial, given that PRPs are the most conserved oral salivary proteins among mammals.


Assuntos
Peptídeos , Processamento de Proteína Pós-Traducional/fisiologia , Proteólise , Proteínas Salivares Ricas em Prolina , Carboxipeptidases/genética , Carboxipeptidases/metabolismo , Humanos , Peptídeos/genética , Peptídeos/metabolismo , Proteínas Salivares Ricas em Prolina/genética , Proteínas Salivares Ricas em Prolina/metabolismo
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