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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.

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

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.

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

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.

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.

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.

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

Graphical abstract, Highlights • DAOY cells have been analyzed by top-down LC-high resolution-MS proteomic platform. • New protein identifications in medulloblastoma cells are reported. • PTMs, isoforms and naturally occurring peptide fragments were identified. • Most of the identified proteins were connected in a biological interacting network. • The data contribute to the further molecular characterization of medulloblastoma., The proteome of the DAOY medulloblastoma cell line has been investigated by an LC–MS top-down platform. This approach, unlike bottom-up ones, allows identifying proteins and peptides in their intact/native forms, disclosing post-translational modifications, proteoforms and naturally occurring peptides. Indeed, 25 out of the 53 proteins identified, were not previously characterized in DAOY cells. Most of them were functionally interconnected, being mainly involved in binding, catalytic and structural activities, and metabolic processes. The top-down approach, applied in this preliminary study, disclosed the presence of several naturally occurring peptide fragments that characterize DAOY cells.

The acid-insoluble salivary proteome obtained by addition of TFA to whole human saliva from adults, preterm and at-term newborns has been analysed by 2-DE in order to evidence differences among the three groups, and integrate data previously obtained on the acid-soluble fraction. 2-DE spots differentially expressed among the three groups were submitted to in-gel tryptic digestion and the peptide mixtures analysed by high resolution HPLC–ESI–MS/MS. By this strategy, we identified 3 over-expressed proteins in at-term newborns with respect to preterm newborns and adults (BPI fold-containing family A member 1, annexin A1, and keratin type 1 cytoskeletal 13), and several over-expressed proteins in adults (fatty acid-binding protein, S100 A6, S100 A7, S100 A9, prolactin-inducible protein, Ig kappa chain, cystatin SN, cystatin S/SA and α-amylase 1). Four spots, already detected but not characterized by other authors in human saliva 2-DE, were attributed to different protein species of S100 A9 (long-type and long-type monophosphorylated, short-type and short-type monophosphorylated) by MS/MS analysis of tryptic peptides and sequential staining of 2-DE gels with Pro-Q Diamond, for specific detection of phosphoproteins, and total protein SYPRO Ruby stain. Significance Differential protein expression analysis of the acid insoluble fraction of saliva from preterm, at-term newborns and adults has been performed in this study by coupling 2-DE analysis and high-resolution tandem mass spectrometry in order to complete the information previously obtained by top-down LC–MS only on the acid-soluble proteome. Several proteins identified in the acid insoluble fraction of both preterm newborn and adult saliva are not of glandular origin, being only prolactin-inducible protein, salivary cystatins, α-amylase and polymeric immunoglobulin receptor exclusive of salivary glands. Three proteins resulted increased in at-term newborns with respect to preterm newborns and adults: BPI fold-containing family A member 1, two proteoforms of annexin A1 and keratin type 1 cytoskeletal 13, while several proteins were significantly increased in adults.

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.

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 ?-Defensins 1-4, Thymosin ?4 and Thymosin ?10, as described in previous works and revealed the presence of other interesting peptides never described before in GCF such as specific fragments of ?-1-antitrypsin, ?-1-antichymotrypsin; fragments of Thymosin ?4 and Thymosin ?10; Fibrinopeptide A and its fragments and Fibrinopeptide B; S100A8 and S100A9, LVV Hemorphin-7 (hemoglobin chain ? fragment), as well as some other peptides deriving from ? and ? 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.

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. SS new components of the family were characterized by top-down liquid chromatography mass spectrometry and tandem-mass platforms, bringing the total number of proteoforms to 109. The new components comprise the three variants P-H S-1 -> A, P-Ko P-36 -> S, and P-Ko A(41) -> 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.

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.

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

An HPLC-ESI-MS analysis of adult human whole saliva evidenced three protein masses (M average 11,487 +/- 2, 11,301 +/- 2 and 22,362 +/- 3 Da) eluting in the 32.5-35.0 mwin range. Treatment in reducing conditions allowed establishing that they are S-derivatives of N-terminal acetylated cystatin B, namely its S-glutathionyl, S-cysteinyl and S-S dimer. The identification was confirmed by high resolution HPLC-ESI-MS-MS experiments on the intact naturally occurring proteins and their tryptic digests. S-unmodified cystatin B is rarely detectable in whole saliva of healthy adults (5 subjects out of 65) and its percentage does not overcome approximately 20% of total cystatin B (11 +/- 9%). In the majority of subjects (60 out of 65) the mean percentages of the S-modified derivatives were S-glutathionyl 53 +/- 13%, S-cysteinyl 15 +/- 5%, S-S 2-mer 32 +/- 13%. Variations of the percentages of these S-modified derivatives of cystatin B could be indicative of oral oxidative stress. As we are aware, this is the first time that S-glutathionylation and S-cysteinylation were described as extensive PTM of a salivary protein and the first time that these PTMs were detected in naturally occurring cystatin B. (C) 2011 Elsevier B.V. All rights reserved.

The increasing attention now paid to the elucidation of human proteome strengthened the development of analytical instruments able to provide reliable proteins and peptides quantitation and characterization in biological fluids and tissues. Emerging from proteomics, clinical proteomics exclusively considers its biomedical applications. It evaluates, often by high-throughput comparative platforms, the protein and peptide variations in body fluids, cells and tissues under different physiological and pathological conditions with the aim of discovering disease biomarkers. Among the available analytical methodologies, mass spectrometry in coupling with liquid chromatography or capillary electrophoresis demonstrated to be the eligible technique for protein detection and identification. This review summarizes the most recent applications of capillary electrophoresis-mass spectrometry to clinical proteomics, focusing on capillary zone electrophoresis separation mode and ESI and MALDI ionizations, which are the most frequently applied capillary electrophoresis-mass spectrometry hyphenated techniques.

Hyperhomocysteinemia is a risk factor for different diseases. Several methods have been developed to analyze homocysteine and the immunometric ones, although expensive, they are in widespread use. A rapid LC-MS/MS method for homocysteine assay has been developed for the application of large clinical chemistry routines. Selected reaction monitoring was performed through the transitions m/z 136.0→90.1 for homocysteine and m/z 140.0→94.0 for the internal standard. ESI was used to generate [H+] adduct ions. Chromatographic isocratic separation was achieved using a strong cation exchange column. The mobile phase was methanol/water (20:80 v/v, containing 0.1% formic acid and 1.5 mmol/L ammonium formate in the water phase) at a flow rate of 0.250 mL/min (35°C). Samples treatment consisted in the reduction with DTT and deproteinization with methanol. Recovery, linearity, LOD, LOQ and total imprecision were evaluated to validate the method. Homocysteine values on 100 serum samples were compared with those obtained by HPLC and immunometric methods. The method is robust, selective and precise in the whole range of values studied. Moreover, low reagent cost and easiness of sample treatment make this method useful, not only for research, but also for routine work.

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.

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 combination of top-down and bottom-up platforms was utilized for the LC-MS proteomic characterization of the intracystic fluid of adamantinomatous craniopharyngioma pediatric brain tumor disease. Proteins and peptides characterization was achieved by high-resolution LC-ESI-LTQ-Orbitrap-MS analysis while low-resolution LC-ESI-IT-MS was applied for the complete screening of the samples and the evaluation of the protein distribution within patients. Top-down analyses were applied to liquid/liquid extracted samples while bottom-up analyses were performed after trypsin digestion of both untreated and pretreated samples. The two proteomic approaches were complementary for the characterization of the proteome of craniopharyngioma intracystic fluid. Proteins and peptides involved in inflammation, mineralization processes and lipid transport were identified, in agreement with the calcium flecks, cholesterol granules and bone residues characteristic of this fluid. Apolipoprotein A-I, A-II, C-I and J, hemoglobin fragments, ubiquitin, α-2-HS-glycoprotein or fetuin A, α-1-antichymotrypsin, vitamin D binding protein, and α-1-acid glycoprotein were characterized. These data could be relevant for the comprehension of the processes involved in the pathogenesis of the disease and the development of the cyst and could contribute to the individuation of therapeutic targets for the reduction of the cyst volume delaying and/or avoiding invasive surgical treatments.

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.

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.

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

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.

During HPLC-ESI-MS/MS analysis of rat submandibular saliva secreted under isoprenaline stimulation, a protein with an experimental [M+H](1+) = 10,544.24 m/z was detected (17.5 ± 0.7 min). The MS/MS fragmentation pattern, manually investigated, allowed establishing an internal sequence in agreement with a DNA-derived sequence of an unknown rat protein coded D3Z9M3 (Swiss-Prot). To match the experimental MS/MS fragmentation pattern and protein mass with theoretical data, the removal from the N terminus of the signal peptide and from the C terminus of three amino acid (a.a.) residues (Arg-Ala-Val) and the cyclization of the N-terminal glutamine in pyroglutamic had to be supposed, resulting in a mature protein of 90 a.a. HPLC-ESI-MS/MS of the trypsin digest ensured 100% sequence coverage. For the high glutamine content (34/90 = 37.8%) we propose to name this protein rat gliadoralin A 1-90. Low amounts of five different isoforms were sporadically detected, which did not significantly change their relative amounts after stimulation. Gliadoralin A is substrate for transglutaminase-2, having Lys 60 and different Gln residues as major determinants for enzyme recognition. In silico investigation of superior structures evidenced that a small part of the protein adopts an α-helical fold, whereas large segments are unfolded, suggesting an unordered conformation.

Thymosin (4) (T-4) is a peptide present in almost any tissue and in extracellular media in mammals, having multiple amazing functions as wound healing, stimulation of angiogenesis, and suppression of inflammation. This study describes its determination in saliva through CE-MS using multiple ions monitoring scan mode by isolating the four most intense multicharged ions present in the MS spectra of the peptide. This scan modality, by reducing the baseline noise and interferences, increases the sensitivity and specificity in biological matrices. The CE-MS separation was optimized by studying different parameters influencing CE analysis, sample injection, and MS ionization, that is, the nebulizer gas flow, the sheath liquid, and BGE composition. The proposed technique can unambiguously identify in short time T-4 in saliva after a very fast and reduced sample pretreatment procedure. The method was validated for quantitation showing linearity of the response in the range 0.25 (lower limit of quantification) to 4 M (average R-2 0.996 +/- 0.005) and intra- and interassay precision and accuracy at three different concentrations with RSD values in the range of 7-16%. It was successfully applied to the analysis of T-4 in whole saliva showing a variable peptide content from individual to individual (in the range of 0.3-1.4 M) and in different days from the same individual. CE-MS in multiple ions monitoring scan mode provides a fast, selective, and economic method requiring only very few microliters of sample.

Thymosin beta(4) sequesters actin by formation of a 1:1 complex. This transient binding in the complex was stabilized by formation of covalent bonds using the cross-linking agents 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and a microbial transglutaminase. The localization of cross-linking sites was determined after separating the products using SDS-PAGE by tryptic in-gel digestion and high-resolution HPLC-ESI-MS. Three cross-linked fragments were identified after chemical cross-linking, indicating three contact sites. Because the cross-linked fragments were detected simultaneously with the corresponding non-cross-linked fragments, the three contact sites were not formed in parallel. K3 of thymosin beta(4) was cross-linked to E167 of actin, K18 or K19 of thymosin beta(4) to one of the first three amino acids of actin (DDE), and S43 of thymosin beta(4) to H40 of actin. The imidazole ring of histidine was proven to be an acyl acceptor for carbodiimide-mediated cross-linking. Molecular modeling proved an extended conformation of thymosin beta(4) along the subdomains 1 to 3 of actin. The enzymatic cross-linking using a microbial transglutaminase led to the formation of three cross-linking sites. Q41 of actin was cross-linked to K19 of thymosin beta(4), and K61 of actin to Q39 of thymosin beta(4). The third cross-linking site was identified between Q41 of actin and Q39 of thymosin beta(4), which are simultaneously cross-linked to K16, K18, or K19 of thymosin beta(4). When both cross-linking reactions are taken together, the complex formation of actin by thymosin beta(4) is more likely to be flexible than rigid and is localized along the subdomains 1 to 3 of actin.

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.

High-performance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) mass -spectrometry (MS) is a relevant technique for the detection and relative quantitation of naturally occurring peptides and proteins. The peptide/protein mass is determined by deconvolution of the ESI-MS spectrum, and the resolution can be better than 1:10,000 with the instruments currently available. Accurate mass measurement, coupled with sufficient resolution, makes it possible to greatly restrict the enormous number of possible molecular formulas that might be represented by a specific molecular mass. As soon as the protein mass has been unequivocally attributed to a specific structure by means of different enzymatic and chemical treatments, the m/z values detected in the ESI spectrum can be utilized to reveal the protein and to perform its relative quantitation, by the extracted ion current (XIC) procedure, in an unlimited number of samples. This chapter describes the HPLC-ESI-MS experimental conditions which allow detecting and quantifying-in human saliva-different S100 proteins and their isoforms.

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

The development of new separation techniques and different mass spectrometry instrumental devices, as well as the great availability of specific reactants, offers ample choice to scientists for carrying out high-throughput proteomic studies and being competitive in the field today. However, the different options available often do not provide comparable results, which can be linked to factors such as the strategy adopted, the nature of the sample and the instrumental availability. In this critical review, the results obtained so far in the study of human saliva by different proteomic approaches will be compared and discussed. © 2012 Expert Reviews Ltd.

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.

RP-HPLC–ESI-MS profile of saliva samples from human preterm newborn showed a protein peak in the elution range 26.6–27.6 min. Deconvolution of ESI-MS spectra revealed the presence of two proteins with average molecular mass (Mav) values of 17,239 ± 3 Da and 18,065 ± 3 Da in 9 samples, with Mav value of 17,239 ± 3 Da in 4 samples and Mav value of 18,065 ± 3 Da in 2 samples. MALDI-TOF-MS analysis of tryptic digest allowed identifying the proteins as two isoforms of small proline-rich protein 3 and cDNA amplification of RNA extracts from oral mucosa, parotid and submandibular gland samples, obtained at fetal autopsy, provided two nucleotide sequences in agreement with those reported in the literature. The two proteins differ for an octapeptide repeat (GCTKVPEP) and the substitution Leu → Val, at position 148 and 140 of the mature form of the 18,065 and 17,239 Da protein, respectively. During maturation the two proteins undergo two post-translational modifications, corresponding to N-terminal acetylation and removal of the initiator methionine. cDNA amplification did not allow to clarify if the proteins found in saliva originated from cellular shedding of the epithelium and/or secretion.

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.