A top-down proteomic approach reveals a salivary protein profile able to classify Parkinson's disease with respect to Alzheimer's disease patients and to healthy controls.

Parkinson's disease (PD) is a complex neurodegenerative disease with motor and non-motor symptoms. Diagnosis is complicated by lack of reliable biomarkers. To individuate peptides and/or proteins with diagnostic potential for early diagnosis, severity and discrimination from similar pathologies, the salivary proteome in 36 PD patients was investigated in comparison with 36 healthy controls (HC) and 35 Alzheimer's disease (AD) patients. A top-down platform based on HPLC-ESI-IT-MS allowed characterizing and quantifying intact peptides, small proteins and their PTMs (overall 51). The three groups showed significantly different protein profiles, PD showed the highest levels of cystatin SA and antileukoproteinase and the lowest of cystatin SN and some statherin proteoforms. HC exhibited the lowest abundance of thymosin ß4, short S100A9, cystatin A, and dimeric cystatin B. AD patients showed the highest abundance of α-defensins and short oxidized S100A9. Moreover, different proteoforms of the same protein, as S-cysteinylated and S-glutathionylated cystatin B, showed opposite trends in the two pathological groups. Statherin, cystatins SA and SN classified accurately PD from HC and AD subjects. α-defensins, histatin 1, oxidized S100A9, and P-B fragments were the best classifying factors between PD and AD patients. Interestingly statherin and thymosin ß4 correlated with defective olfactory functions in PD patients. All these outcomes highlighted implications of specific proteoforms involved in the innate-immune response and inflammation regulation at oral and systemic level, suggesting a possible panel of molecular and clinical markers suitable to recognize subjects affected by PD.

Top-Down Proteomics Detection of Potential Salivary Biomarkers for Autoimmune Liver Diseases Classification.

(1) Autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC) are autoimmune liver diseases characterized by chronic hepatic inflammation and progressive liver fibrosis. The possible use of saliva as a diagnostic tool has been explored in several oral and systemic diseases. The use of proteomics for personalized medicine is a rapidly emerging field. (2) Salivary proteomic data of 36 healthy controls (HCs), 36 AIH and 36 PBC patients, obtained by liquid chromatography/mass spectrometry top-down pipeline, were analyzed by multiple Mann-Whitney test, Kendall correlation, Random Forest (RF) analysis and Linear Discriminant Analysis (LDA); (3) Mann-Whitney tests provided indications on the panel of differentially expressed salivary proteins and peptides, namely cystatin A, statherin, histatin 3, histatin 5 and histatin 6, which were elevated in AIH patients with respect to both HCs and PBC patients, while S100A12, S100A9 short, cystatin S1, S2, SN and C showed varied levels in PBC with respect to HCs and/or AIH patients. RF analysis evidenced a panel of salivary proteins/peptides able to classify with good accuracy PBC vs. HCs (83.3%), AIH vs. HCs (79.9%) and PBC vs. AIH (80.2%); (4) RF appears to be an attractive machine-learning tool suited for classification of AIH and PBC based on their different salivary proteomic profiles.

The Post-Translational Modifications of Human Salivary Peptides and Proteins Evidenced by Top-Down Platforms.

In this review, we extensively describe the main post-translational modifications that give rise to the multiple proteoforms characterized to date in the human salivary proteome and their potential role. Most of the data reported were obtained by our group in over twenty-five years of research carried out on human saliva mainly by applying a top-down strategy. In the beginning, we describe the products generated by proteolytic cleavages, which can occur before and after secretion. In this section, the most relevant families of salivary proteins are also described. Next, we report the current information concerning the human salivary phospho-proteome and the limited news available on sulfo-proteomes. Three sections are dedicated to the description of glycation and enzymatic glycosylation. Citrullination and N- and C-terminal post-translational modifications (PTMs) and miscellaneous other modifications are described in the last two sections. Results highlighting the variation in the level of some proteoforms in local or systemic pathologies are also reviewed throughout the sections of the manuscript to underline the impact and relevance of this information for the development of new diagnostic biomarkers useful in clinical practice.

Combined Salivary Proteome Profiling and Machine Learning Analysis Provides Insight into Molecular Signature for Autoimmune Liver Diseases Classification.

Autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC) are autoimmune liver diseases that target the liver and have a wide spectrum of presentation. A global overview of quantitative variations on the salivary proteome in presence of these two pathologies is investigated in this study. The acid-insoluble salivary fraction of AIH and PBC patients, and healthy controls (HCs), was analyzed using a gel-based bottom-up proteomic approach combined with a robust machine learning statistical analysis of the dataset. The abundance of Arginase, Junction plakoglobin, Desmoplakin, Hexokinase-3 and Desmocollin-1 decreased, while that of BPI fold-containing family A member 2 increased in AIHp compared to HCs; the abundance of Gelsolin, CD14, Tumor-associated calcium signal transducer 2, Clusterin, Heterogeneous nuclear ribonucleoproteins A2/B1, Cofilin-1 and BPI fold-containing family B member 2 increased in PBCp compared to HCs. The abundance of Hornerin decreased in both AIHp and PBCp with respect to HCs and provided an area under the ROC curve of 0.939. Machine learning analysis confirmed the feasibility of the salivary proteome to discriminate groups of subjects based on AIH or PBC occurrence as previously suggested by our group. The topology-based functional enrichment analysis performed on these potential salivary biomarkers highlights an enrichment of terms mostly related to the immune system, but also with a strong involvement in liver fibrosis process and with antimicrobial activity.

Salivary Cystatin D Interactome in Patients with Systemic Mastocytosis: An Exploratory Study.

Mastocytosis, a rare blood disorder characterized by the proliferation of clonal abnormal mast cells, has a variegated clinical spectrum and diagnosis is often difficult and delayed. Recently we proposed the cathepsin inhibitor cystatin D-R26 as a salivary candidate biomarker of systemic mastocytosis (SM). Its C26 variant is able to form multiprotein complexes (mPCs) and since protein-protein interactions (PPIs) are crucial for studying disease pathogenesis, potential markers, and therapeutic targets, we aimed to define the protein composition of the salivary cystatin D-C26 interactome associated with SM. An exploratory affinity purification-mass spectrometry method was applied on pooled salivary samples from SM patients, SM patient subgroups with and without cutaneous symptoms (SM+C and SM-C), and healthy controls (Ctrls). Interactors specifically detected in Ctrls were found to be implicated in networks associated with cell and tissue homeostasis, innate system, endopeptidase regulation, and antimicrobial protection. Interactors distinctive of SM-C patients participate to PPI networks related to glucose metabolism, protein S-nitrosylation, antibacterial humoral response, and neutrophil degranulation, while interactors specific to SM+C were mainly associated with epithelial and keratinocyte differentiation, cytoskeleton rearrangement, and immune response pathways. Proteins sensitive to redox changes, as well as proteins with immunomodulatory properties and activating mast cells, were identified in patients; many of them were involved directly in cytoskeleton rearrangement, a process crucial for mast cell activation. Although preliminary, these results demonstrate that PPI alterations of the cystatin D-C26 interactome are associated with SM and provide a basis for future investigations based on quantitative proteomic analysis and immune validation.

A Catalog of Coding Sequence Variations in Salivary Proteins' Genes Occurring during Recent Human Evolution.

Saliva houses over 2000 proteins and peptides with poorly clarified functions, including proline-rich proteins, statherin, P-B peptides, histatins, cystatins, and amylases. Their genes are poorly conserved across related species, reflecting an evolutionary adaptation. We searched the nucleotide substitutions fixed in these salivary proteins' gene loci in modern humans compared with ancient hominins. We mapped 3472 sequence variants/nucleotide substitutions in coding, noncoding, and 5'-3' untranslated regions. Despite most of the detected variations being within noncoding regions, the frequency of coding variations was far higher than the general rate found throughout the genome. Among the various missense substitutions, specific substitutions detected in PRB1 and PRB2 genes were responsible for the introduction/abrogation of consensus sequences recognized by convertase enzymes that cleave the protein precursors. Overall, these changes that occurred during the recent human evolution might have generated novel functional features and/or different expression ratios among the various components of the salivary proteome. This may have influenced the homeostasis of the oral cavity environment, possibly conditioning the eating habits of modern humans. However, fixed nucleotide changes in modern humans represented only 7.3% of all the substitutions reported in this study, and no signs of evolutionary pressure or adaptative introgression from archaic hominins were found on the tested genes.

Saliva, a bodily fluid with recognized and potential diagnostic applications.

Human whole saliva is a bodily fluid that can be obtained easily by noninvasive techniques. Specimens can be collected by the patient also at home in order to monitor health status and variations of several analytes of clinical interest. The contributions to whole saliva include secretions from salivary glands and, among others, from the gingival crevicular fluid that derives from the epithelial mucosa. Therefore, saliva is currently a relevant diagnostic fluid for many substances, including steroids, nonpeptide hormones, therapeutic drugs, and drugs of abuse. This review at first briefly describes the different contributions to whole saliva. A section illustrates the procedures for the collection, handling, and storage of salivary specimens. Another section describes the present use of whole saliva for diagnostic purposes and its specific utilization for the diagnosis of several local and systemic diseases. The final sections illustrate the future opportunities offered by various not conventional techniques with a focus on the most recent -omic investigations. It describes the various issues that have to be taken into account to avoid false positives and negatives, such as the strength of the experimental plan, the adequacy of the number of samples under study, and the proper choice of controls.

Differences in Salivary Proteins as a Function of PROP Taster Status and Gender in Normal Weight and Obese Subjects.

Taste plays an important role in processes such as food choices, nutrition status and health. Salivary proteins contribute to taste sensitivity. Taste reduction has been associated with obesity. Gender influences the obesity predisposition and the genetic ability to perceive the bitterness of 6-n-propylthiouracil (PROP), oral marker for food preferences and consumption. We investigated variations in the profile of salivary proteome, analyzed by HPLC-ESI-MS, between sixty-one normal weight subjects (NW) and fifty-seven subjects with obesity (OB), based on gender and PROP sensitivity. Results showed variations of taste-related salivary proteins between NW and OB, which were differently associated with gender and PROP sensitivity. High levels of Ps-1, II-2 and IB-1 proteins belonging to basic proline rich proteins (bPRPs) and PRP-1 protein belonging to acid proline rich proteins (aPRPs) were found in OB males, who showed a lower body mass index (BMI) than OB females. High levels of Ps-1 protein and Cystatin SN (Cyst SN) were found in OB non-tasters, who had lower BMI than OB super-tasters. These new insights on the role of salivary proteins as a factor driving the specific weight gain of OB females and super-tasters, suggest the use of specific proteins as a strategic tool modifying taste responses related to eating behavior.

Cryptides: latent peptides everywhere.

Proteomic surveys with top-down platforms are today revealing thousands of naturally occurring fragments of bigger proteins. Some of them have not functional meaning because they derive from pathways responsible for protein degradation, but many have specific functions, often completely different from that one of the parent proteins. These peptides encrypted in the protein sequence are nowadays called cryptides. They are frequent in the animal and plant kingdoms and represent a new interesting -omic field of investigation. To point out how much widespread is their presence, we describe here the most studied cryptides from very common sources such as serum albumin, immunoglobulins, hemoglobin, and from saliva and milk proteins. Given its vastness, it is unfeasible to cover the topic exhaustively, therefore only several selected examples of cryptides from other sources are thereafter reported. Demanding is the development of new -omic platforms for the functional screening of new cryptides, which could provide suggestion for peptides and peptido-mimetics with variegate fields of application.

Mapping of Transglutaminase-2 Sites of Human Salivary Small Basic Proline-Rich Proteins by HPLC-High-Resolution ESI-MS/MS.

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.

Top-Down Proteomics of Human Saliva Discloses Significant Variations of the Protein Profile in Patients with Mastocytosis.

Mastocytosis is a myeloproliferative neoplasm causing abnormal clonal mast cell accumulation in different tissues, such as skin and bone marrow. A cutaneous subtype (CM) is distinguished from a systemic one (SM); SM patients can be grouped into SM with (SM+C) or without (SM-C) additional cutaneous lesions, and their classification is often challenging. This study was purposed to highlight variations in the salivary proteome of patients with different mastocytosis subtypes and compared to healthy controls. A top-down proteomics approach coupled to a label-free quantitation revealed salivary profiles in patients different from those of controls and a down-regulation of peptides/proteins involved in the mouth homeostasis and defense, such as statherin, histatins, and acidic proline-rich proteins (aPRPs), and in innate immunity and inflammation, such as the cathepsin inhibitors, suggesting a systemic condition associated with an exacerbated inflammatory state. The up-regulation of antileukoproteinase and S100A8 suggested a protective role against the disease status. The two SM forms were distinguished by the lower levels of truncated forms of aPRPs, statherin, P-B peptide, and cystatin D and the higher levels of thymosin ß4 and α-defensins 1 and 4 in SM-C patients with respect to SM+C. Data are available via ProteomeXchange with identifier PXD017759.

Proteomic Analysis of the Acid-Insoluble Fraction of Whole Saliva from Patients Affected by Different Forms of Non-histaminergic Angioedema.

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.

RP-HPLC-ESI-IT Mass Spectrometry Reveals Significant Variations of the Human Salivary Protein Profile Associated with Predominantly Antibody Deficiencies.

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

Enrichments of post-translational modifications in proteomic 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.

Extensive Characterization of the Human Salivary Basic Proline-Rich Protein Family by Top-Down Mass Spectrometry.

Human basic proline-rich proteins and basic glycosylated proline-rich proteins, encoded by the polymorphic PRB1-4 genes and expressed only in parotid glands, are the most complex family of adult salivary proteins. The family includes 11 parent peptides/proteins and more than 6 parent glycosylated proteins, but a high number of proteoforms with rather similar structures derive from polymorphisms and post-translational modifications. 55 new components of the family were characterized by top-down liquid chromatography-mass spectrometry and tandem-mass platforms, bringing the total number of proteoforms to 109. The new components comprise the three variants P-H S1 → A, P-Ko P36 → S, and P-Ko A41 → S and several of their naturally occurring proteolytic fragments. The paper represents an updated reference for the peptides included in the heterogeneous family of proteins encoded by PRB1/PRB4. MS data are available via ProteomeXchange with the identifier PXD009813.

Marked Differences in the Submandibular Salivary Proteome between Sardinian Alcohol-Preferring and Sardinian Alcohol-Non Preferring Rats Revealed by an Integrated Top-Down-Bottom-Up Proteomic Platform.

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.

Salivary Cystatins: Exploring New Post-Translational Modifications and Polymorphisms by Top-Down High-Resolution Mass Spectrometry.

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.

Characterization of the cell penetrating properties of a human salivary proline-rich peptide.

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 proline-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-ß-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.

Proteomics applied to pediatric medicine: opportunities and challenges.

INTRODUCTION: Care in pediatrics often refers to treatments directed to adults. However, childhood is a specific life period, with molecular pathways connected to development and thereby it requires distinctive considerations and special treatments under disease. Proteomics can help to elucidate the molecular mechanisms underlying the human development and disease onset in pediatric age and this review is devoted to underline the results recently obtained in the field. AREAS COVERED: The contribution of proteomics to the characterization of physiological modifications occurring during human development is presented. The proteomic studies carried out to elucidate the molecular mechanisms underlying different pediatric pathologies and to discover new markers for early diagnosis and prognosis of disease, comprising genetic and systemic pathologies, sepsis and pediatric oncology are thereafter reported. The investigations concerning milk composition in human and farm mammals are also presented. Finally, the chances offered by the integration of different -omic platforms are discussed. Expert commentary: The growing utilization of holistic technologies such as proteomics, metabolomics and microbiomics will allow, in the near future, to define at the molecular level the complexity of human development and related diseases, with great benefit for future generations.

N- and O-linked glycosylation site profiling of the human basic salivary proline-rich protein 3M.

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.