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Background: Nimotuzumab is a humanized anti-epidermal growth factor receptor(EGFR) monoclonal antibody, nowadays used for tumour immunochemotherapy. This study aimedto label the conjugate DOTA-nimotuzumab with yttrium-90, in order to provide a β- emitting radioimmunoconjugate(90Y-DOTA-nimotuzumab) potentially useful to assess the feasibility of anew radio-guided surgery approach. Methods: The synthesis of 90Y-DOTA-nimotuzumab was performed in two days. Nimotuzumabwas conjugated with a 50-fold excess of DOTA and then labelled with 90Y3+. The 90Y-DOTA-nimotuzumabpreparation was optimized considering several parameters such as pH, temperature and reactionvolume. Moreover, the 90Y-DOTA-nimotuzumab stability was evaluated in human plasma. Results: The radioimmunoconjugate 90Y-DOTA-nimotuzumab was obtained with a radiochemicalpurity greater than 96%, and showed a good stability at 20°C as well as at 37°C in human plasma. Conclusions: The optimized conditions for a mild and easy preparation of 90Y-DOTA-nimotuzumabjoined to a promising stability under physiological conditions suggest to propose this radioimmunoconjugateas a potential diagnostic radiopharmaceutical for β- radio-guided surgery.

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.

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

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 (P32and A32variants) 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: Q29of P-H, Q37of P-D, Q21of II-2, Q41of P-C, and Q37of 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.

Human basic proline-rich proteins and basic glycosylated proline-rich proteins, encoded by the polymorphic PRB1–4genes 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.

ABSTRACTObjectives: We reevaluated a lyophilized sample of thymosin fraction 5, stored for 37 years at room temperature, by high-resolution mass spectrometry in terms of stability and yet uncharacterized polypeptides that could be biological important substances.Methods: A top–down proteomic platform based on high-performance liquid chromatography (HPLC) coupled to high-resolution LTQ-Orbitrap mass spectrometry (MS) was applied to molecular characterization of polypeptides present in thymosin fraction 5.Results: We detected more than 100 monoisotopic masses corresponding to thymosin β4 and truncated forms of ubiquitin, prothymosin α, thymosin β4, and thymosin β9. Additionally, we discovered a new polypeptide present in thymosin fraction 5 and identified it as intact SH3 domain-binding glutamic acid-rich-like protein 3.Conclusion: In spite of the well-known proteolytic processes inherent to the preparation of thymosin fraction 5, still uncharacterized polypeptides as well as truncated forms of already well-known thymosins are present in fraction 5 after long-term storage. Therefore, continuing characterization of thymosin fraction 5 is even nowadays highly promising.

Background: Infectious etiologies contribute to 15% of male factor infertility. Enterococcus faecalis (E. faecalis) is commonly identified in semen culture of infertile men and it is associated with significantly poorer semen quality. Objective: Aim of this study was to identify new seminal biomarkers for the male tract infection by E. faecalis, using proteomic profiling, in order to understand the effect of E. faecalis on the physiopathology of male reproduction. Methods: We included in the study ten patients seeking medical care for primary infertility with prostate-vesicular-epidydimitis and with microbiological analysis on semen and/or prostatic secretions positive for E. faecalis. Ten fertile men have been enrolled as a control group in the protocol. An aliquot of each seminal plasma was subjected to an in-solution digestion protocol and analyzed using an Ultimate 3000 RSLCnano HPLC apparatus coupled to a LTQ Orbitrap Elite mass spectrometer. Results: Eight proteins have not been identified in the group of controls and have been observed in a remarkable proportion of patients, mainly involved in immune system activity (CD177, Swiprosin-1 and 2-oxoglutarate dehydrogenase). Arylsulfatase has been identified in the group of controls and was absent in all patients with infection. Three proteins (TIMP-1, WFDC domain protein 2 and Carboxypeptidase E) have been observed significantly different in patients versus controls, mainly related with inflammation. Conclusions: This is the first application of MS-based proteomics aimed to reveal an array of proteins in the seminal plasma and reflecting the effect of the infection by E. faecalis on semen composition.

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.

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

In the present study, we investigated whether thymosin β (Tβ) in saliva and in minor salivary glands is differentially expressed in patients with primary Sjögren’s syndrome (pSS) and patients with autoimmune diseases (systemic sclerosis [SSc], systemic lupus erythematosus [SLE], and rheumatoid arthritis [RA], with and without sicca syndrome [ss]). Saliva specimens of nine patients with pSS, seven with ss/SSc, seven with ss/SLE, seven with ss/RA, seven with SSc, seven with SLE, and seven with RA, as well as ten healthy subjects, were analyzed using a high-performance liquid chromatograph coupled with a mass spectrometer equipped with an electrospray ionization source to investigate the presence and levels of Tβ4, Tβ4sulfoxide, and Tβ10. Immunostaining for Tβ4and Tβ10was performed on minor salivary glands of patients with pSS and ss. Tβ4levels were statistically higher in patients with pSS with respect to the other subgroups. Tβ10was detectable in 66.7 % of patients with pSS and in 42.8 % of those with ss/SSc, while Tβ4sulfoxide was detectable in 44.4 % of patients with pSS and in 42.9 % of those with ss/SSc. Tβ10and Tβ4sulfoxide were not detectable in patients without associated ss and in healthy control subjects. Regarding thymosin immunostaining, all patients had immunoreactivity for Tβ10, and a comparable distribution pattern in the four different subgroups of patients was observed. Tβ4immunoreactivity was present in patients with ss/SSc and those with ss/SLE, while it was completely absent in patients with pSS and those with ss/RA. Our data show that higher salivary Tβ expression characterizes patients with pSS, while Tβ4sulfoxide and Tβ10salivary expression was selectively present in patients with sicca symptoms. Moreover, at the immunohistochemical level in patients with pSS, minor salivary glands showed a peculiar pattern characterized by immunostaining for Tβ10in acinar cells in the absence of any reactivity for Tβ4. These findings, taken together, suggest a different role for Tβ4and Tβ10in patients with pSS who have ss and other autoimmune disease.

Objectives: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.Methods:A top–down proteomic platform based on high-performance liquid chromatography (HPLC) coupled to high-resolution LTQ-Orbitrap mass spectrometry (MS) was applied to the characterization of naturally occurring peptides.Results: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 Tβ4and Tβ10together with other proteolytic fragments. The sulfoxide derivative of both Tβ4and Tβ10and the acetylated derivatives at lysine residues of Tβ4were also characterized. Different proteoforms of prothymosin α, parathymosin α, thymosin α1and thymosin α11together with diverse proteolytic fragments were identified too.Conclusion:The clinical and prognostic significance and the origin of these proteoforms have to be deeply investigated.

Introduction:The purpose of our work was to identify unknown interaction partners of thymosin β4(Tβ4). It was suggested that Tβ4could be an antifibrotic drug for treatment of liver fibrogenesis, because Tβ4prevents the platelet-derived growth factor-BB (PDGF-BB)-induced activation of hepatic stellate cells (HSCs). Very little information is available how Tβ4counteracts the PDGF-BB-induced activation of HSCs. We propose the hypothesis that Tβ4could bind directly to PDGF-BB and thereby reduce the concentration of free PDGF-BB available for binding to the PDGF-β receptor.Methods:To prove our suggestion of a direct interaction between Tβ4and PDGF-BB, we carried out chemical as well as photochemical cross-linking experiments between the two pure proteins in vitro.Results:We identified an interaction between Tβ4and 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 vitrosystem, 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.Conclusion:Direct interaction of Tβ4with extracellular factors should be considered as a potential mechanism to explain the pleiotropic effects of β-thymosins.

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 PRH2locus and glycosylated basic proline-rich proteins encoded by PRB3locus 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 PRH1locus 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.

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.

Thymosin beta 4 (Tβ4) is a ubiquitous peptide that plays pivotal roles in the cytoskeletal system and in cell differentiation. Recently, a role for Tβ4 has been proposed in experimental and human carcinogenesis, including gastrointestinal cancer. This study was aimed at evaluating the relationship between Tβ4 immunoreactivity and the initial steps of carcinogenesis.

Background: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.Methods: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 a-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.Results:Twenty-five studied neonates were divided into two groups: Mn-positive group and Mn-negative group. Levels of a-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 a-defensins 1–4 and S100A8 were observed.Conclusion: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 a-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 α-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.

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. MSanalyses on the intact glycoproteins before and after N‐deglycosylation with PNGase Fand high‐resolution MS/MSsequencing by LTQOrbitrap XLof 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.

ObjectiveThymosin β-4 (Tβ4) is a ubiquitous peptide that plays pivotal roles in the cytoskeletal system and in cell differentiation during embryogenesis. Recently, a role for Tβ4has been proposed in experimental and human carcinogenesis. This study was aimed at evaluating the correlation between Tβ4immunoractivity and colorectal cancer, with particular attemption to tumor cells undergoing epithelial-mesenchymal transition.Methods and Results86 intestinal biopsies were retrospectively analyzed including 76 colorectal adenocarcinomas with evident features of epithelial-mesenchymal transition, and 10 samples of normal colorectal mucosa. Paraffin sections were immunostained for Tβ4and for E-cadherin. Total RNA was isolated from frozen specimens obtained, at surgery, from the normal colon mucosa, the deeper regions and the superficial tumor regions in four cases of colon cancer. Tβ4immunoreactivity was detected in the vast majority (59/76) of colon carcinomas, showing a patchy distribution, with well differentiated areas significantly more reactive than the less differentiated tumor zones. We also noted a zonal pattern in the majority of tumors, characterized by a progressive increase in immunostaining for Tβ4from the superficial toward the deepest tumor regions. The strongest expression for Tβ4was frequently detected in invading tumor cells with features of epithelial-mesenchymal transition. The increase in reactivity for Tβ4matched with a progressive decrease in E-cadherin expression in invading cancer cells. At mRNA level, the differences in Tβ4expression between the surrounding colon mucosa and the tumors samples were not significant.ConclusionsOur data show that Tβ4is expressed in the majority of colon cancers, with preferential immunoreactivity in deep tumor regions. The preferential expression of the peptide and the increase in intensity of the immunostaining at the invasion front suggests a possible link between the peptide and the process of epithelial mesenchymal transition, suggesting a role for Tβ4in colorectal cancer invasion and metastasis.

The acidic soluble fraction of whole saliva of type 1 diabetic children was analyzed by reversed phase (RP)11The abbreviations used are:aPRPssalivary acidic proline-rich phosphoproteinsbPRPsbasic salivary proline-rich proteinsP-Bproline-rich peptide P-BRPreversed phaseIgimmunoglobulinXICextracted ion currentPRPproline-rich proteinsHst-1histatin-1.–HPLC-ESI-MS and compared with that of sex- and age-matched control subjects. Salivary acidic proline-rich phosphoproteins (aPRP), histatins, α-defensins, salivary cystatins, statherin, proline-rich peptide P-B (P-B), beta-thymosins, S100A8 and S100A9*(S100A9* corresponds to S100A9 vairant lacking the first four amino acids), as well some naturally occurring peptides derived from salivary acidic proline-rich phosphoproteins, histatins, statherin, and P-B peptide, were detected and quantified on the basis of the extracted ion current peak area. The level of phosphorylation of salivary acidic proline-rich phosphoproteins, histatin-1 (Hst-1), statherin and S100A9* and the percentage of truncated forms of salivary acidic proline-rich phosphoproteins was also determined in the two groups. The study revealed that statherin, proline-rich peptide P-B, P-C peptide, and histatins, were significantly less concentrated in saliva of diabetic subjects than in controls, while concentration of α-defensins 1, 2 and 4 and S100A9* was higher. The low concentration of P-C peptide was paralleled by high levels of some of its fragments. On the whole, the study highlighted the severe impairment of the repertoire of peptides involved in the safeguard of the oral cavity in children who have diabetes, as well as an higher concentration of the proinflammatory mediator S100A9* with respect to healthy children.

The acidic soluble fraction of whole saliva of type 1 diabetic children was analyzed by reversed phase (RP)1–HPLC-ESI-MS and compared with that of sex- and age-matched control subjects. Salivary acidic proline-rich phosphoproteins (aPRP), histatins, α-defensins, salivary cystatins, statherin, proline-rich peptide P-B (P-B), beta-thymosins, S100A8 and S100A9*(S100A9* corresponds to S100A9 vairant lacking the first four amino acids), as well some naturally occurring peptides derived from salivary acidic proline-rich phosphoproteins, histatins, statherin, and P-B peptide, were detected and quantified on the basis of the extracted ion current peak area. The level of phosphorylation of salivary acidic proline-rich phosphoproteins, histatin-1 (Hst-1), statherin and S100A9* and the percentage of truncated forms of salivary acidic proline-rich phosphoproteins was also determined in the two groups. The study revealed that statherin, proline-rich peptide P-B, P-C peptide, and histatins, were significantly less concentrated in saliva of diabetic subjects than in controls, while concentration of α-defensins 1, 2 and 4 and S100A9* was higher. The low concentration of P-C peptide was paralleled by high levels of some of its fragments. On the whole, the study highlighted the severe impairment of the repertoire of peptides involved in the safeguard of the oral cavity in children who have diabetes, as well as an higher concentration of the proinflammatory mediator S100A9* with respect to healthy children.

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.

The presence of a-defensins in bronchoalveolar lavage fluid (BALF) was investigated in a cohort of preterm newborns with gestational age (GA) =30 wk. Specimens were collected during the first week of life from 24 preterm neonates mechanically ventilated. The studied population was divided into two groups: pneumonia group of nine neonates suffering from pulmonary infection (GA: 26.1 ± 2.1 wk; birth weight: 787.4 ± 309.9 g), with or without associated bloodstream infection, and nonpneumonia group of 15 neonates (GA: 27.7 ± 2.0 wk; birth weight: 1019.0 ± 319.8 g). BALF culture was positive for CONS (n = 5), Staphylococcus aureus (n = 1), and Candida spp (n = 3). BALF samples were analyzed by HPLC-electrospray Ionization-mass spectrometer. The a-defensins 1–4 concentration, absolute and differential white cells count were measured. Relative amounts of a-defensins 1–4 and the absolute number of neutrophils were found significantly higher in the pneumonia group with respect to the nonpneumonia group (p < 0.05). Moreover, positive significant correlations between the number of neutrophils and the a-defensins 1–3 levels were observed. In conclusion, our data show that preterm newborns, also at the lower GA, are able to produce a-defensins, underlining that their innate defense system is already active before the at-term delivery date.

The presence of -defensins in bronchoalveolar lavage fluid (BALF) was investigated in a cohort of preterm newborns with gestational age (GA) ≤30 wk. Specimens were collected during the first week of life from 24 preterm neonates mechanically ventilated. The studied population was divided into two groups: pneumonia group of nine neonates suffering from pulmonary infection (GA: 26.1 ± 2.1 wk; birth weight: 787.4 ± 309.9 g), with or without associated bloodstream infection, and nonpneumonia group of 15 neonates (GA: 27.7 ± 2.0 wk; birth weight: 1019.0 ± 319.8 g). BALF culture was positive for CONS (n5), Staphylococcus aureus(n1), and Candidaspp (n3). BALF samples were analyzed by HPLC-electrospray Ionization-mass spectrometer. The -defensins 1–4 concentration, absolute and differential white cells count were measured. Relative amounts of -defensins 1–4 and the absolute number of neutrophils were found significantly higher in the pneumonia group with respect to the nonpneumonia group (p< 0.05). Moreover, positive significant correlations between the number of neutrophils and the -defensins 1–3 levels were observed. In conclusion, our data show that preterm newborns, also at the lower GA, are able to produce -defensins, underlining that their innate defense system is already active before the at-term delivery date.

Listeria monocytogenesis frequently found as a contaminant in raw and ready‐to‐eat foods. The ability of L. monocytogenesto multiply at refrigeration temperatures and to grow in a wide range of pH values is of particular concern for food safety. According to the European Union regulation on microbiological criteria for foodstuffs, L. monocytogenesmust be absent in some categories of ready‐to‐eat foods. The standard microbiological method for L. monocytogenesdetection in foods (ISO 11290‐1: 1996 (ISO, International Organization for Standardization)) is cost and time consuming. Developments of rapid, cost‐effective and automated diagnostic methods to detect food‐borne pathogens in foods continue to be a major concern for the industry and public health. The aim of this study was the development of a rapid, sensitive and specific molecular detection method for L. monocytogenes. To this purpose, we have applied a capillary electrophoresis method to a PCR protocol (PCR‐EES (EES, experion automated electrophoresis system)) for detecting L. monocytogenesin food. In particular, a microfluidic chip‐based automated electrophoresis system (experion automated electrophoresis system, Bio‐Rad Laboratories, USA) was used for the rapid and automatic analysis of the amplicons. Fifty naturally contaminated samples were analysed with this method and the results were compared with those obtained with ISO method. Moreover, the microfluidic chip‐based automated electrophoresis system was compared with classical gel electrophoresis (PCR‐CGE). The results showed that after 24 h of culture enrichment, the PCR‐EES showed a relative accuracy of 100% with ISO, while using PCR‐CGE decreased it down to 96%. After 48 h of enrichment, both PCR‐EES and PCR‐CGE showed an accuracy of 100% with ISO.

Two peptides (MW 1211.7 and 928.5 Da) were detected by RP–HPLC–ESI–MS analysis of parotid saliva secreted upon continuous parasympathetic stimulation. The peptide with the higher mass (PSPFr‐A) corresponded to the N‐terminal dodecapeptide (Fragment 1–12) of rat parotid secretory protein (PSP), while the peptide with the lower mass (PSPFr‐B) corresponded to the 4–12 fragment of the same protein. During stimulation, the PSPFr‐A secretion increased, while the PSPFr‐B secretion decreased (HPLC–ESI–MS). In the presence of cycloheximide, PSPFr‐A was not demonstrated, while the PSPFr‐B secretion decreased. In the presence of aprotinin, the PSPFr‐B secretion was almost abolished, while the PSPFr‐A secretion increased to higher levels than those observed in the absence of the inhibitor. In vitroperfusion, with artificial solution, of stimulated rat parotid glands excluded that the fragments were derived from the circulation. Neither peptide occurred in enriched granule preparations from unstimulated glands. The results suggest that at least two pathways – granular and vesicular – are responsible for the generation of the two peptides. PSPFr‐A is the first cleavage product in both pathways. PRPFr‐B is probably generated from granular PSPFr‐A only and, at the end of the granule mediated pathway, by the action of an enzyme of the serine protease class.

Thymosin β4(Tβ4), its sulfoxide, and thymosin β10 (Tβ10) were detected in human saliva and identified by different strategies based on RP HPLC coupled to electrospray multidimensional IT MS. Tβ4 was almost always detected in whole saliva, its sulfoxide sporadically, Tβ10rarely. Tβ4was undetectable in parotid saliva and less concentrated in submandibular/sublingual saliva than in whole saliva. Analysis of gingival crevicular fluid revealed high relative amounts of Tβ4, Tβ4sulfoxide, and Tβ10in all the samples. Tβ4mean concentration was 200 times higher in crevicular fluid (20 μmol/L, N= 9) than in whole saliva (0.1 μmol/L, N= 9). Crevicular fluid concentration of Tβ4 (ca. 5% represented by its sulfoxide) and β10 significantly correlated (r= 0.856; N= 9), and their ratio was about 5. A significant correlation was also observed between Tβ4 concentrations in whole saliva and gingival crevicular fluid (r= 0.738;N= 9). Immunohistochemical analysis of the major salivary glands showed that immunoreactivity for Tβ4is restricted to ductal cells, with minor degree of focal positivity in some acinar cells. On the whole, results indicate that gingival sulcus is a main, although not the sole, source for oral Tβ4 and Tβ10.

Glucose‐6‐phosphate dehydrogenase (G6PD) deficiency is one of the most well‐known human genetic defects, identified in more than 400 million individuals in the world. To date, no commercial kits are available for the mutation screening of this disease. Seventy G6PD‐deficient Italian individuals admitted to the Laboratory of Clinical Molecular Biology of Hospital “Agostino Gemelli” of Rome were screened for the most frequent Italian mutations, by means of allele‐specific PCR, followed by restriction fragment length electrophoresis. The present study compares two techniques for the identification of restriction patterns: agarose gel electrophoresis versus Experion system. When the first screening was negative, the entire G6PD gene was sequenced using the ABI 3100 Avant Instrumentation. The G6PD variants identified and their frequencies were the following: G6PD Mediterranean (75.7%), G6PD Seattle (7.1%), G6PD A–202 + 376 (7.1%), and G6PD Cassano (2.8%). In addition, we identified by direct sequencing two new mutations, namely Buenos Aires and Rignano. With the Experion method, the size band determination was more accurate than that obtained by gel electrophoresis. The Experion system resulted as a valid, easy, and reproducible diagnostic method for the screening of G6PD mutation as compared with the agarose electrophoretic analysis.

This review briefly depicts several salient points of the current status of knowledge on salivary peptidoma. It outlines the intrinsic difficulties in its characterization connected to different factors of variability, such as: i) the high genetic polymorphisms, complicated by individual insertions/deletions and alternative splicing; ii) complex post‐translational maturations comprehending different proteolytic cleavages, glycosylation, phosphorylation and sulfation processes; iii) physiological variations and different contributions to the whole. Moreover, several technological and analytical problems and pitfalls that had to be surmounted during our studies focussed on the extensive qualitative and quantitative characterization of salivary peptidoma and mainly based on LC‐MS analyses of intact naturally occurring peptides are here described. The hope is that the information provided might be helpful to other groups engaged on the analysis of saliva or other body fluids for clinical applications.

To elucidate the localization of post-translational modifications of different classes of human salivary proteins and peptides (acidic and basic proline-rich proteins (PRPs), Histatins, Statherin, P-B peptide, and “S type” Cystatins) a comparative reversed phase HPLC-ESI-MS analysis on intact proteins of enriched granule preparations from parotid and submandibular glands as well as parotid, submandibular/sublingual (Sm/Sl), and whole saliva was performed. The main results of this study indicate the following. (i) Phosphorylation of all salivary peptides, sulfation of Histatin 1, proteolytic cleavages of acidic and precursor basic PRPs occur before granule storage. (ii) In agreement with previous studies, basic PRPs are secreted by the parotid gland only, whereas all isoforms of acidic PRPs (aPRPs) are secreted by both parotid and Sm/Sl glands. (iii) Phosphorylation levels of aPRPs, Histatin 1, and Statherin are higher in the parotid gland, whereas the extent of cleavage of aPRP is higher in Sm/Sl glands. (iv) O-Sulfation of tyrosines of Histatin 1 is a post-translational modification specific for the submandibular gland. (v) The concentration of Histatin 3, Histatin 5, and Histatin 6, but not Histatin 1, is higher in parotid saliva. (vi) Histatin 3 is submitted to the first proteolytic cleavage (generating Histatins 6 and 5) during granule maturation, and it occurs to the same relative extent in both glands. (vii) The proteolytic cleavages of Histatin 5 and 6, generating a cascade of Histatin 3 fragments, take place after granule secretion and are more extensive in parotid secretion. (viii) Basic PRPs are cleaved in the oral cavity by unknown peptidases, generating various small proline-rich peptides. (ix) C-terminal removal from Statherin is more extensive in parotid saliva. (x) P-B peptide is secreted by both glands, and its relative quantity is higher in submandibular/sublingual secretion. (xi) In agreement with previous studies, S type Cystatins are mainly the product of Sm/Sl glands.

To elucidate the localization of post-translational modifications of different classes of human salivary proteins and peptides (acidic and basic proline-rich proteins (PRPs), Histatins, Statherin, P-B peptide, and "S type" Cystatins) a comparative reversed phase HPLC-ESI-MS analysis on intact proteins of enriched granule preparations from parotid and submandibular glands as well as parotid, submandibular/sublingual (Sm/Sl), and whole saliva was performed. The main results of this study indicate the following. (i) Phosphorylation of all salivary peptides, sulfation of Histatin 1, proteolytic cleavages of acidic and precursor basic PRPs occur before granule storage. (ii) In agreement with previous studies, basic PRPs are secreted by the parotid gland only, whereas all isoforms of acidic PRPs (aPRPs) are secreted by both parotid and Sm/Sl glands. (iii) Phosphorylation levels of aPRPs, Histatin 1, and Statherin are higher in the parotid gland, whereas the extent of cleavage of aPRP is higher in Sm/Sl glands. (iv) O-Sulfation of tyrosines of Histatin 1 is a post-translational modification specific for the submandibular gland. (v) The concentration of Histatin 3, Histatin 5, and Histatin 6, but not Histatin 1, is higher in parotid saliva. (vi) Histatin 3 is submitted to the first proteolytic cleavage (generating Histatins 6 and 5) during granule maturation, and it occurs to the same relative extent in both glands. (vii) The proteolytic cleavages of Histatin 5 and 6, generating a cascade of Histatin 3 fragments, take place after granule secretion and are more extensive in parotid secretion. (viii) Basic PRPs are cleaved in the oral cavity by unknown peptidases, generating various small proline-rich peptides. (ix) C-terminal removal from Statherin is more extensive in parotid saliva. (x) P-B peptide is secreted by both glands, and its relative quantity is higher in submandibular/sublingual secretion. (xi) In agreement with previous studies, S type Cystatins are mainly the product of Sm/Sl glands.

Basic proline‐rich proteins (bPRPs) are a class of proteins widely present in saliva of humans and other mammals. They are synthesized as preproproteins and enzymatically cleaved into small peptides before secretion from the salivary glands. Recently, we characterized two proline‐rich peptides (SP‐A and SP‐B) in parotid secretory granules of pig (Sus Scrofa) that are derived from three isoforms of a PRP proprotein (Swiss‐Prot data bank:Q95JC9‐1, Q95JC9‐2 andQ95JC9‐3). Together the coding regions for SP‐A and SP‐B, which are repeated many times, account for 52–70% of the coding regions of the PRP proproteins. This study was undertaken to identify peptides encoded by unassigned regions of the PRP proproteins. RP‐HPLC‐ESI‐IT‐MS analysis of enriched granule preparations from pig parotid glands by two different analytical strategies identified ten new proline‐rich peptides derived from the three proproteins. Together with the coding regions for SP‐A and SP‐B already identified it was possible to assign 68–75% of the proproteins coding regions. The peptide sequences indicated a number of unusual proteolytic cleavage sites suggesting the presence of unknown proprotein convertases.

In the present study the analytical potential of HPLC–MS/MS was utilized for the structural characterization of a post‐translational modification of statherin. Human salivary statherin (Mav5380.0 ± 0.3 Da) is transformed by the action of transglutaminase 2 into a cyclic derivative with an average molecular mass of 5363.0 ± 0.3 Da. The intra‐molecular bridge is generated by the loss of an ammonia molecule between the unique lone‐pair donating nucleophile Lys‐6 and one acceptor among the seven glutamine residues of statherin. Digestion of the cyclic derivative with chymotrypsin, proteinase K, and carboxypeptidase Y, monitored by HPLC–electrospray ionization‐ion trap‐mass spectrometric analysis, demonstrated that cyclization involved almost specifically Gln‐37 (>95%), with the percentage of Gln‐39 implicated in the cross‐linking being less than 5%. The main derivative was named cyclo‐statherin Q‐37. Guinea pig transglutaminase 2 showed high affinity for statherin in vitro(Km= 0.65 ± 0.06 μM). Cyclo‐statherin was detectedin vivo by HPLC‐electrospray ionization ion trap‐mass spectrometry analysis of whole human saliva and it accounted for about 1% of total statherin. Detection of cyclo‐statherin in whole saliva is suggestive of a putative role of this molecule in the formation of the “oral protein pellicle”.

The myoglobin of the polychaete annelid Ophelia bicornis was isolated, purified to homogeneity and characterized. The primary structure, obtained from cDNA and protein sequencing, consists of 139 amino acid residues. The alignment with other globin sequences showed that O. bicornis myoglobin misses the pre-A helix and the first six residues of the A helix. The presence of a PheB10-GlnE7 haem distal residue pair is in agreement with the measured oxygen affinity (P50=0.85 mmHg; 1 mmHg=0.133 kPa) and the only slightly higher autoxidation rate constant (0.28 h−1) with respect to that of the sperm whale myoglobin mutant E7 His→Gln (0.21 h−1) and to elephant myoglobin (0.1 h−1). Oxygen-binding co-operativity was found to be absent under all the examined experimental conditions. The resistance of O. bicornis myoglobin towards autoxidation seems to confirm the important role of part of the A helix in the stability of the globin. The higher pKa of the acid–alkaline ferric transition of O. bicornis with respect to Asian elephant myoglobin, as well as the higher absorbance ratio of its ferric form to the oxy form measured in the Soret region (γmet/γoxy) with respect to that of the African elephant myoglobin, suggested a stronger interaction between the distal glutamine and the water molecule at the sixth co-ordinate position.

Haemoglobins from mammals of sub-Arctic and Arctic species, as well as fetal human Hb, are all characterized by a significantly lower ΔH of oxygenation compared with the majority of mammalian haemoglobins from temperate species (exceptions are represented by some cold-resistant species, such as cow, horse and pig). This has been interpreted as an adaptive mechanism of great importance from a physiological point of view. To date, the molecular basis of this thermodynamic characteristic is still not known. In the present study, we show that binding of extra chloride (with respect to adult human Hb) ions to Hb would significantly contribute to lowering the overall heat of oxygenation, thus providing a molecular basis for the low effect of temperature on the oxygenation–deoxygenation cycle. To this aim, the oxygen binding properties of bovine Hb, bear (Ursus arctos) Hb and horse Hb, which are representative of this series of haemoglobins, have been studied with special regard to the effect of heterotropic ligands, such as organic phosphates (namely 2,3-diphosphoglycerate) and chloride. Functional results are consistent with a mechanism for ligand binding that involves an additional binding site for chloride ion. Analysis of computational chemistry results, obtained by the GRID program, further confirm the hypothesis that the reason for the lower ΔH of oxygenation is mainly due to an increase in the number of the oxygen-linked chloride-binding sites.

The Conger conger (conger eel) haemoglobin (Hb) system is made of three components, one of which, the so-called cathodic Hb, representing approx. 20% of the total pigment, has been purified and characterized from both a structural and functional point of view. Stripped Hb showed a reverse Bohr effect, high oxygen affinity and slightly low cooperativity in the absence of any effector. Addition of saturating GTP strongly influences the pH dependence of the oxygen affinity, since the reverse Bohr effect, observed under stripped conditions, is converted into a small normal Bohr effect. A further investigation of the GTP effect on oxygen affinity, carried out by fitting its titration curve, demonstrated the presence of two independent binding sites. Therefore, on the basis of the amino acid sequence of the α- and β-chains, which have been determined, a computer modelling study has been performed. The data suggest that C. conger cathodic Hb may bind organic phosphates at two distinct binding sites located along the central cavity of the tetramer by hydrogen bonds and/or electrostatic interactions with amino acid residues of both chains, which have been identified. Among these residues, the two Lys-α(G6) (where the letter refers to the haemoglobin helix and the number to the amino acid position in the helix) appear to have a key role in the GTP movement from the external binding region to the internal central cavity of the tetrameric molecule.

Sulfate transport by band-3 protein in adult human erythrocytes was shown to be modulated by oxygen pressure. In particular, a higher transport activity was measured under high oxygen pressure than at low one (0.0242±0.0073 vs. 0.0074±0.0010 min). Other factors, such as magnesium ions and orthovanadate, which can indirectly affect the binding properties of the cytoplasmic domain of band 3 (cdb3), influence significantly the anion exchanger activity. No effect of oxygen pressure on sulfate transport was found in chicken erythrocytes, which may be related to their lacking the cdb3 binding site. These findings are fully consistent with a molecular mechanism where the oxygen-linked transition of hemoglobin (T→R) could play a key role in the regulation of anion exchanger activity.

This study describes the capillary electrophoresis detection of the weak interaction occurring between the negative form of 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES^–) and nicotinamide-adenine dinucleotide (NAD⁺). The ion-paired complex is formed probably by 1 molecule of each reactant and it is sensitive to the applied electric field, since it was found that the value of the apparent association constant decreases as a function of the field applied. The association constant at zero field was obtained by extrapolation of the values determined at different applied field strengths and was found to be 2.0×10³ M^–1. The peak convolution observed at the detection window was representative of a dissociation process that started at the beginning of the separation. The rate of the field-induced dissociation corresponded to 6.4×10^–10 mol·cm·L^–1·V^–1·s^–1. Due to the complete absence of detectable signal shifts in ¹H-NMR experiments, the interaction should be ascribed to the negative sulpho-moiety of HEPES^– and to the positive charge of the NAD⁺ pyrimidine ring. To our knowledge, this is the first example of a “secondary Wien effect” observed at low field.

This study describes the capillary electrophoresis detection of the weak interaction occurring between the negative form of 4‐(2‐hydroxyethyl)‐1‐piperazine‐ethanesulfonic acid (HEPES–) and nicotinamide‐adenine dinucleotide (NAD+). The ion‐paired complex is formed probably by 1 molecule of each reactant and it is sensitive to the applied electric field, since it was found that the value of the apparent association constant decreases as a function of the field applied. The association constant at zero field was obtained by extrapolation of the values determined at different applied field strengths and was found to be 2.0×103M–1. The peak convolution observed at the detection window was representative of a dissociation process that started at the beginning of the separation. The rate of the field‐induced dissociation corresponded to 6.4×10–10mol·cm·L–1·V–1·s–1. Due to the complete absence of detectable signal shifts in 1H‐NMR experiments, the interaction should be ascribed to the negative sulpho‐moiety of HEPES–and to the positive charge of the NAD+pyrimidine ring. To our knowledge, this is the first example of a “secondary Wien effect” observed at low field.

Ergothioneine (ESH) is a low-molecular-mass thiol present in millimolar concentrations in a limited number of tissues, including erythrocytes, kidney, seminal fluid and liver; however, its biological function is still unclear. In the present study we investigated the role of ESH in the catabolism of S-nitrosoglutathione (GSNO). The results show that: (1) GSNO decomposition is strongly influenced by ESH (k′′ = 0.178±0.032M−1·s−1); (2) ammonia is the main nitrogen-containing compound generated by the reaction; and (3) nitrite is practically absent under both aerobic and anaerobic conditions. These findings are markedly different from those reported for the GSH-induced decomposition of GSNO, in which the nitrogen-containing end products are nitrite, ammonia and nitrous oxide (N2O) under aerobic conditions but nitrite, ammonia, nitric oxide (NO) and small quantities of hydroxylamine under anaerobic conditions. Considering the high concentration of ESH in specific cells, the reaction with GSNO should be considered as an important molecular event occurring in the cell.

A nucleotide pyrophosphatase (EC 3.6.1.9) was purified to homogeneity from lentil seedlings. The enzyme is a single polypeptide chain of 75 ± 2 kDa that exhibits hydrolytic activities toward pyrophosphate linkages of several substrates. Reduced and oxidized forms of NAD(P) were shown to be hydrolyzed to nicotinamide mononucleotide and AMP. Other dinucleotides such as FAD and dinucleoside oligophosphates were hydrolyzed as well, but with lower efficiency. Pyrophosphatase activity was increased in the presence of divalent cations such as Ca2+, Mg2+, and Mn2+, whereas Cu2+, Zn2+, and Ni2+ions inhibited this activity. The active site in the enzyme was not defined, but histidine residue(s) seemed to be crucial for the enzymatic activity.