Salivary Proteome and Intact N-Glycopeptides Analysis Reveal Specific Signatures in Periodontitis.

Periodontitis is a prevalent oral inflammatory disease that can result in tooth loss and is closely linked to type 2 diabetes (T2D). In this study, we analyzed the salivary proteome and intact N-glycopeptides (IGPs) of individuals with mild-moderate, severe, aggressive periodontitis, and periodontitis with T2D, including those treated with antidiabetic drugs, to identify specific signatures associated with the disease. Our results revealed that salivary proteins and glycoproteins were altered in all periodontitis groups (PRIDE ID: 1-20230612-72345), with fucose- and sialic acid-containing N-glycans showing the greatest increase. Additionally, differentially expressed proteins were classified into 9 clusters, including those that were increased in all periodontitis groups and those that were only altered in certain types of periodontitis. Interestingly, treatment with antidiabetic drugs reversed many of the changes observed in the salivary proteome and IGPs in T2D-related periodontitis, suggesting a potential therapeutic approach for managing periodontitis in patients with T2D. Consistent with MS/MS results, the expression of salivary IGHA2 and Fucα1-3/6GlcNAc (AAL) was significantly increased in MP. These findings provide new insights into the pathogenesis of periodontitis and highlight the potential of salivary biomarkers for diagnosis, prognosis, and monitoring of disease progression and treatment response.

Secreted protein markers in oral squamous cell carcinoma (OSCC).

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a main cause of oral cancer mortality and morbidity in central south Asia. To improve the clinical outcome of OSCC patients, detection markers are needed, which are preferably non-invasive and thus independent of a tissue biopsy. METHODS: In the present study, we aimed to identify robust candidate protein biomarkers for non-invasive OSCC diagnosis. To this end, we measured the global protein profiles of OSCC tissue lysates to matched normal adjacent mucosa samples (n = 14) and the secretomes of nine HNSCC cell lines using LC-MS/MS-based proteomics. RESULTS: A total of 5123 tissue proteins were identified, of which 205 were robustly up- regulated (p-value < 0.01, fold change > + 2) in OSCC-tissues compared to normal adjacent tissues. The biological process "Secretion" was highly enriched in this set of proteins. Other upregulated biological pathways included "Unfolded Protein Response", "Spliceosomal complex assembly", "Protein localization to endosome" and "Interferon Gamma Response". Transcription factor analysis implicated Creb3L1, ESRRA, YY, ELF2, STAT1 and XBP as potential regulators. Of the 205 upregulated tissue proteins, 132 were identified in the cancer cell line secretomes, underscoring their potential use as non-invasive biofluid markers. To further prioritize our candidate markers for non-invasive OSCC detection, we integrated our data with public biofluid datasets including OSCC saliva, yielding 25 candidate markers for further study. CONCLUSIONS: We identified several key proteins and processes that are associated with OSCC tissues, underscoring the importance of altered secretion. Cancer-associated OSCC secretome proteins present in saliva have potential to be used as novel non-invasive biomarkers for the diagnosis of OSCC.

The potential impact of salivary peptides in periodontitis.

Periodontitis is a complex immune-inflammatory condition characterized by the disruption of the periodontal ligament and subsequent formation of periodontal pockets, and by alveolar bone loss, often resulting in tooth loss. A myriad of factors, namely, genetic, metabolic, immunological, and inflammatory, is associated with progression of periodontitis. Periodontitis is also associated with systemic conditions such as neoplastic disorders, obesity, and diabetes. The current diagnosis of this disease relies on clinical measurements such as clinical attachment loss and probing depth, which have poor precision due to patient, operator and probe-related factors. Thus, there is a need to develop reliable, objective, and reproducible biomarkers for early diagnosis of periodontitis. In this regard, saliva, with contributions from the gingival crevicular fluid, holds great potential. However, most of the information on biomarkers of periodontium-related salivary proteins has come from studies on the molecular pathogenesis of periodontitis. In periodontitis, a more holistic approach, such as the use of -omics technologies, for biomarker discovery, is needed. Herein, we review the biomarkers proposed to date for the assessment of periodontitis, with emphasis on the role of salivary peptides in periodontitis and their assessment by high-throughput saliva proteomics. We also discuss the challenges pertaining to the identification of new periodontitis biomarkers in saliva.

Human and Nonhuman Primate Lineage-Specific Footprints in the Salivary Proteome.

Proteins in saliva are needed for preprocessing food in the mouth, maintenance of tooth mineralization, and protection from microbial pathogens. Novel insights into human lineage-specific functions of salivary proteins and clues to their involvement in human disease can be gained through evolutionary studies, as recently shown for salivary amylase AMY1 and salivary agglutinin DMBT1/gp340. However, the entirety of proteins in saliva, the salivary proteome, has not yet been investigated from an evolutionary perspective. Here, we compared the proteomes of human saliva and the saliva of our closest extant evolutionary relatives, chimpanzees and gorillas, using macaques as an outgroup, with the aim to uncover features in saliva protein composition that are unique to each species. We found that humans produce a waterier saliva, containing less than half total protein than great apes and Old World monkeys. For all major salivary proteins in humans, we could identify counterparts in chimpanzee and gorilla saliva. However, we discovered unique protein profiles in saliva of humans that were distinct from those of nonhuman primates. These findings open up the possibility that dietary differences and pathogenic pressures may have shaped a distinct salivary proteome in the human lineage.

Comparative salivary proteomics analysis of children with and without dental caries using the iTRAQ/MRM approach.

BACKGROUND: Dental caries is a major worldwide oral disease afflicting a large proportion of children. As an important host factor of caries susceptibility, saliva plays a significant role in the occurrence and development of caries. The aim of the present study was to characterize the healthy and cariogenic salivary proteome and determine the changes in salivary protein expression of children with varying degrees of active caries, also to establish salivary proteome profiles with a potential therapeutic use against dental caries. METHODS: In this study, unstimulated saliva samples were collected from 30 children (age 10-12 years) with no dental caries (NDC, n = 10), low dental caries (LDC, n = 10), and high dental caries (HDC, n = 10). Salivary proteins were extracted, reduced, alkylated, trypsin digested and labeled with isobaric tags for relative and absolute quantitation, and then they were analyzed with GO annotation, biological pathway analysis, hierarchical clustering analysis, and protein-protein interaction analysis. Targeted verifications were then performed using multiple reaction monitoring mass spectrometry. RESULTS: A total of 244 differentially expressed proteins annotated with GO annotation in biological processes, cellular component and molecular function were identified in comparisons among children with varying degrees of active caries. A number of caries-related proteins as well as pathways were identified in this study. As compared with caries-free children, the most significantly enriched pathways involved by the up-regulated proteins in LDC and HDC were the ubiquitin mediated proteolysis pathway and African trypanosomiasis pathway, respectively. Subsequently, we selected 53 target proteins with differential expression in different comparisons, including mucin 7, mucin 5B, histatin 1, cystatin S and cystatin SN, basic salivary proline rich protein 2, for further verification using MRM assays. Protein-protein interaction analysis of these proteins revealed complex protein interaction networks, indicating synergistic action of salivary proteins in caries resistance or cariogenicity. CONCLUSIONS: Overall, our results afford new insight into the salivary proteome of children with dental caries. These findings might have bright prospect in future in developing novel biomimetic peptides with preventive and therapeutic benefits for childhood caries.

iTRAQ-based quantitative analysis of age-specific variations in salivary proteome of caries-susceptible individuals.

BACKGROUND: Human saliva is a protein-rich, easily accessible source of potential biomarkers for the diagnosis of oral and systemic diseases. However, little is known about the changes in salivary proteome associated with aging of patients with dental caries. Here, we applied isobaric tags for relative and absolute quantitation (iTRAQ) in combination with multiple reaction monitoring mass spectrometry (MRM-MS) to characterize the salivary proteome profiles of subjects of different ages, presenting with and without caries, with the aim of identifying age-related biomarkers for dental caries. METHODS: Unstimulated whole saliva samples were collected from 40 caries-free and caries-susceptible young adults and elderly individuals. Salivary proteins were extracted, reduced, alkylated, digested with trypsin and then analyzed using iTRAQ-coupled LC-MS/MS, followed by GO annotation, biological pathway analysis, hierarchical clustering analysis, and protein-protein interaction analysis. Candidate verification was then conducted using MRM-MS. RESULTS: Among 658 salivary proteins identified using tandem mass spectrometry, 435 proteins exhibited altered expression patterns in different age groups with and without caries. Of these proteins, 96 displayed age-specific changes among caries-susceptible adults and elderly individuals, and were mainly associated with salivary secretion pathway, while 110 age-specific proteins were identified among healthy individuals. It was found that the age factor caused significant variations and played an important role in both healthy and cariogenic salivary proteomes. Subsequently, a total of 136 target proteins with complex protein-protein interactions, including 14 age-specific proteins associated with caries, were further successfully validated using MRM analysis. Moreover, non-age-specific proteins (histatin-1 and BPI fold-containing family B member 1) were verified to be important candidate biomarkers for common dental caries. CONCLUSIONS: Our proteomic analysis performed using the discovery-through-verification pipeline revealed distinct variations caused by age factor in both healthy and cariogenic salivary proteomes, highlighting the significance of age in the great potential of saliva for caries diagnosis and biomarker discovery.

Saliva proteome profiling reveals potential salivary biomarkers for detection of oral cavity squamous cell carcinoma.

Oral cavity squamous cell carcinoma (OSCC), which is frequently associated with poor prognosis and mortality, is a leading cause of cancer-related death worldwide. Discovery of body fluid accessible biomarkers is needed to improve OSCC screening. To this end, we profiled proteomes of saliva from the healthy volunteers, the individuals with oral potentially malignant disorders (OPMD), and the OSCC patients by means of SDS-PAGE coupled with LC-MS/MS. In the control, the OPMD, and the OSCC groups, 958, 845, and 1030 salivary proteins were detected, respectively. With spectral counting-based label-free quantification, 22 overexpressed salivary proteins were identified in the OSCC group compared with the healthy controls and the OPMD individuals. Among them, resistin (RETN) was subjected to further validation with an independent cohort using ELISA. The data confirmed that the salivary RETN levels in the OSCC patients were significantly higher than that in the healthy or in the OPMD group. Moreover, the elevated levels of salivary RETN were highly correlated with late-stage primary tumors, advanced overall stage, and lymph-node metastasis. Our results not only reveal that profiling of saliva proteome is feasible for discovery of OSCC biomarkers, but also identify RETN as a potential salivary biomarker for OSCC detection.

Towards further defining the proteome of mouse saliva.

BACKGROUND: Knowledge of the mouse salivary proteome is not well documented and as a result, very limited. Currently, several salivary proteins remain unidentified and for some others, their function yet to be determined. The goal of the present study is to utilize mass spectrometry analysis to widen our knowledge of mouse salivary proteins, and through extensive database searches, provide further insight into the array of proteins that can be found in saliva. A comprehensive mouse salivary proteome will also facilitate the development of mouse models to study specific biomarkers of many human diseases. RESULTS: Individual saliva samples were collected from male and female mice, and later pooled according to sex. Two pools of saliva from female mice (2 samples/pool) and 2 pools of saliva from male mice were used for analysis utilizing high performance liquid chromatograph mass spectrometry (nano-RPLC-MS/MS). The resulting datasets identified 345 proteins: 174 proteins were represented in saliva obtained from both sexes, as well as 82 others that were more female specific and 89 that were more male specific. Of these sex linked proteins, twelve were identified as exclusively sex-limited; 10 unique to males and 2 unique to females. Functional analysis of the 345 proteins identified 128 proteins with catalytic activity characteristics; indicative of proteins involved in digestion, and 35 proteins associated with stress response, host defense, and wound healing functions. Submission of the list of 345 proteins to the BioMart data mining tool in the Ensembl database further allowed us to identify a total of 283 orthologous human genes, of which, 131 proteins were recently reported to be present in the human salivary proteome. CONCLUSIONS: The present study is the most comprehensive list to date of the proteins that constitute the mouse salivary proteome. The data presented can serve as a useful resource for identifying potentially useful biomarkers of human health and disease.

Morphology and Proteome Characterization of the Salivary Glands of the Western Chinch Bug (Hemiptera: Blissidae).

The western chinch bug, Blissus occiduus Barber, is a serious pest of buffalograss, Buchloe dactyloides (Nuttall) due to physical and chemical damage caused during the feeding process. Although previous work has investigated the feeding behaviors of chinch bugs in the Blissus complex, no study to date has explored salivary gland morphology and the associated salivary complex of this insect. Whole and sectioned B. occiduus salivary glands were visualized using light and scanning electron microscopy to determine overall structure and cell types of the salivary glands and their individual lobes. Microscopy revealed a pair of trilobed principal glands and a pair of tubular accessory glands of differing cellular types. To link structure with function, the salivary gland proteome was characterized using liquid chromatography tandem mass spectrometry. The salivary proteome analysis resulted in B. occiduus sequences matching 228 nonhomologous protein sequences of the pea aphid, Acyrthosiphon pisum (Harris), with many specific to the proteins present in the salivary proteome of A. pisum. A number of sequences were assigned the molecular function of hydrolase and oxido-reductase activity, with one specific protein sequence revealing a peroxidase-like function. This is the first study to characterize the salivary proteome of B. occiduus and the first of any species in the family Blissidae.

Comparative analyses of salivary proteins from three aphid species.

Saliva is a critical biochemical interface between aphids and their host plants; however, the biochemical nature and physiological functions of aphid saliva proteins are not fully elucidated. In this study we used a multidisciplinary proteomics approach combining liquid chromatography-electrospray ionization tandem mass spectrometry and two-dimensional differential in-gel electrophoresis/matrix-assisted laser desorption/ionization time-of-flight/mass spectrometry to compare the salivary proteins from three aphid species including Acyrthosiphon pisum, Megoura viciae and Myzus persicae. Comparative analyses revealed variability among aphid salivary proteomes. Among the proteins that varied, 22% were related to DNA-binding, 19% were related to GTP-binding, and 19% had oxidoreductase activity. In addition, we identified a peroxiredoxin enzyme and an ATP-binding protein that may be involved in the modulation of plant defences. Knowledge of salivary components and how they vary among aphid species may reveal how aphids target plant processes and how the aphid and host plant interact.

Saliva-microbe interactions and salivary gland dysfunction.

Adequate salivary secretion is crucial to both oral and general health, since it provides a complex milieu for support of the microbial populations of the mouth, while at the same time containing antimicrobial products that help control these microbial populations. This paper summarizes several aspects of salivary component function, gland secretion mechanisms, and immunopathogenesis as related to oral health and disease. Salivary components mediate microbial attachment to oral surfaces, and also interact with planktonic microbial surfaces to facilitate agglutination and elimination of pathogens from the oral cavity. Adhesive interactions are often mediated by lectin-like bacterial proteins that bind to glycan motifs on salivary glycoproteins. An important salivary antimicrobial protein is histatin 5 (Hst 5), which shows potent and selective antifungal activity and also susceptibility to proteolytic degradation. Coupling of Hst 5 with the carrier molecule spermidine significantly enhanced killing of C. albicans and resistance to proteolytic degradation, compared with the parent peptide. Loss of salivary secretion may be caused by disorders such as Sjögren's syndrome (SS) or ectodermal dysplasia, or may be a side-effect of radiation therapy. Two new approaches to the treatment of salivary gland dysfunction include the use of resolvins and the creation of differentiated acinar structures to construct an artificial salivary gland. B-cells contribute to the pathogenesis of SS by releasing cytokines and autoantibodies and by influencing T-cell differentiation. CXCL13, a potent B-cell chemokine associated with autoimmune diseases, is elevated locally and systemically in SS and may represent a novel biomarker or therapeutic target in the management and treatment of SS.

Changes in the saliva proteome analysed by gel-proteomics in horses diagnosed with equine gastric ulcer syndrome (EGUS) at diagnosis and after successful treatment.

Equine gastric ulcer syndrome (EGUS) is currently one of the more frequent diseases in horses. We aimed to identify changes in the salivary proteome in horses with EGUS at diagnosis and after successful treatment by using gel proteomics. Saliva samples were collected from nine horses with EGUS before and after treatment and nine matched healthy controls. SDS-PAGE (1DE) and two-dimensional gel electrophoresis (2DE) were performed, and significantly different protein bands and spots were identified by mass spectrometry. Horses with EGUS had increases in proteins such as adenosine deaminase (ADA), triosephosphate isomerase, keratins and immuno-globulin heavy constant mu and decreases in carbonic anhydrase (CA), albumin and prolactin-induced protein. These changes would indicate various physiopathological mechanisms involved in this disease, such as the activation of the immune system, decreased stomach defence mechanisms and inflammation. The treated horses presented lower expression levels of thioredoxin (TRX) after a successful treatment, in proteomics analysis and also measured with a commercially available ELISA kit. Overall, horses with EGUS have protein changes in their saliva when measured with gel proteomics compared with healthy horses, and they also showed changes after successful treatment. These proteins could be potential biomarkers for detection and monitoring treatment response in EGUS.

Umami Altering Salivary Proteome: A Study across a Sensitivity Spectrum on Subjects.

This study delved into the relationship between umami taste sensitivity (UTS) and variations in the salivary proteome among 12 healthy nonsmokers utilizing 4D data-independent acquisition-based proteomics. By assessing UTS through monosodium l-glutamate (MSG) detection thresholds, we discovered notable differences: individuals with high UTS detected umami at significantly lower MSG concentrations (0.20 ± 0.12 mM) compared to their low UTS counterparts (2.51 ± 1.21 mM). Both groups showed an upregulation of the S100A1 protein under MSG stimulation, indicating a potent biochemical response to umami stimuli. The high UTS group exhibited enhanced metabolic pathways including those for amino acid, lipid, and organic acid biosynthesis, essential for maintaining taste receptor functionality and enhancing signal transduction. This group also demonstrated increased activity in cytochrome P450 enzymes and ribonucleoprotein complexes, suggesting a readiness to manage metabolic challenges and optimize umami perception. In contrast, the low UTS group showed adaptive mechanisms, possibly through modulation of receptor availability and function, with an upregulation of structural and ribosomal proteins that may support taste receptor production and turnover. These findings suggest that varying biological mechanisms underpin differences in umami perception, which could significantly influence dietary preferences and nutritional outcomes, highlighting the intricate interplay of genetic, physiological, and metabolic factors in taste sensitivity.

Changes in the Saliva Proteome of Pigs with Diarrhoea Caused by Escherichia coli.

Escherichia coli represents the main cause of diarrhoea in pigs. Saliva can provide information about the pathophysiology of diseases and be a source of biomarkers. We aimed to identify changes in the salivary proteome of pigs with diarrhoea caused by E. coli. Saliva samples were collected from 10 pigs with this disease and 10 matched healthy controls. SDS-PAGE (1DE) and two-dimensional gel electrophoresis (2DE) were performed, and significantly different protein bands and spots were identified by mass spectrometry. For validation, adenosine deaminase (ADA) was measured in 28 healthy and 28 diseased pigs. In 1DE, increases in lipocalin and IgA bands were observed for diseased pigs, whereas bands containing proteins such as odorant-binding protein and/or prolactin-inducible protein presented decreased concentrations. Two-dimensional gel electrophoresis (2DE) results showed that saliva from E. coli animals presented higher expression levels of lipocalin, ADA, IgA and albumin peptides, being ADA activity increased in the diseased pigs in the validation study. Spots containing alpha-amylase, carbonic anhydrase VI, and whole albumin were decreased in diseased animals. Overall, pigs with diarrhoea caused by E. coli have changes in proteins in their saliva related to various pathophysiological mechanisms such as inflammation and immune function and could potentially be biomarkers of this disease.

Salivary Proteomics Reveals Significant Changes in Relation to Alzheimer's Disease and Aging.

BACKGROUND: Aging is a risk factor for several pathologies as Alzheimer's disease (AD). Great interest exists, therefore, in discovering diagnostic biomarkers and indicators discriminating biological aging and health status. To this aim, omic investigations of biological matrices, as saliva, whose sampling is easy and non-invasive, offer great potential. OBJECTIVE: Investigate the salivary proteome through a statistical comparison of the proteomic data by several approaches to highlight quali-/quantitative variations associated specifically either to aging or to AD occurrence, and, thus, able to classify the subjects. METHODS: Salivary proteomic data of healthy controls under-70 (adults) and over-70 (elderly) years old, and over-70 AD patients, obtained by liquid chromatography/mass spectrometry, were analyzed by multiple Mann-Whitney test, Kendall correlation, and Random-Forest (RF) analysis. RESULTS: Almost all the investigated proteins/peptides significantly decreased in relation to aging in elderly subjects, with or without AD, in comparison with adults. AD subjects exhibited the highest levels of α-defensins, thymosin ß4, cystatin B, S100A8 and A9. Correlation tests also highlighted age/disease associated differences. RF analysis individuated quali-/quantitative variations in 20 components, as oxidized S100A8 and S100A9, α-defensin 3, P-B peptide, able to classify with great accuracy the subjects into the three groups. CONCLUSION: The findings demonstrated a strong change of the salivary protein profile in relation to the aging. Potential biomarkers candidates of AD were individuated in peptides/proteins involved in antimicrobial defense, innate immune system, inflammation, and in oxidative stress. RF analysis revealed the feasibility of the salivary proteome to discriminate groups of subjects based on age and health status.

Impact of the Glycemic Level on the Salivary Proteome of Middle-Aged and Elderly People With Type 2 Diabetes Mellitus: An Observational Study.

Type 2 diabetes mellitus (T2DM) is an increasing global public health concern, but its impact on the salivary proteome is still unclear. To evaluate the effect of glycemic levels in middle-aged and elderly individuals with T2DM on salivary proteomics, we compared the differences by liquid chromatography tandem mass spectrometry (LC-MS/MS). Unstimulated whole saliva samples from 8 T2DM patients with good glycemic control (G group, HbA1c <6.5%) and 16 patients with poor control (P group, HbA1c ≥6.5%) were analyzed by LC-MS/MS in the data-independent acquisition mode (Clinical register number: ChiCTR1900023582.). After functional annotation, cluster analysis and receiver operating characteristic (ROC) curve analysis were carried out to screen and evaluate candidate proteins. A total of 5,721 proteins were quantified, while 40 proteins differed significantly. In the P group, proteins involved in oxidative stress-related processes were upregulated, whereas proteins related to salivary secretion were downregulated. The combination of thioredoxin domain-containing protein 17, zymogen granule protein 16B, and FAM3 metabolism regulating signaling molecule D yielded an area under the curve of 0.917 which showed a robust ability to distinguish the P and G groups. In conclusion, poorly controlled hyperglycemia may affect salivary proteins through various pathways, including oxidative stress and glandular secretion. Furthermore, the differentially expressed proteins, especially the three proteins with the best differentiation, might serve as an anchor point for the further study of hyperglycemia and oral diseases.

Salivary proteome of a Neotropical primate: potential roles in host defense and oral food perception.

BACKGROUND: Saliva contains a very complex mixture of proteins for defense against microbiological pathogens and for oral food perception. Howler monkeys are Neotropical primates that can consume a mostly leaf diet. They are well known to thrive in highly disturbed habitats where they may cope with a diversity of dietary challenges and infection risks. We aimed to describe the salivary proteome of howlers to contribute to better understanding of their physiology. METHODS: We analyzed the salivary proteins of wild black howler monkeys (Alouatta pigra), by SDS-PAGE-1-D and Nano LC-MS/MS and categorized them by their function involved in host defense and oral food perception. RESULTS: Our proteomic analysis identified 156 proteins in howler saliva including a number of host defense peptides that are the first line of defense in mammals, such as defensin, cathelicidin, dermcidin, and lactotransferrin, and proteins with anti-bacterial, anti-fungal, and anti-viral capacity, such as IgA, IgG, IgM, BPI, salivary heat shock 70 kDa protein, beta-2-microbulin, and protein S-100. We also identified key proteins necessary for taste perception, including salivary carbonic anhydrase VI, cystatin D, IgA, and fatty acid-binding protein. Proteins to detect astringent foods were identifying, including four members of cystatins (A, B, C and D), lactoperoxidase, and histidine-rich proteins. No chitinase and amylase were identified as would be expected because howlers do not eat insects and little starch. These findings provide basic information to future studies in oral biology, ingestive physiology, and physiological ecology of mammals and non-human primates.

On the characterisation of the porcine gland-specific salivary proteome.

To expand the knowledge on the porcine salivary proteome, secretions from the three major salivary glands were collected from anaesthetised piglets. Pilocarpine and isoproterenol were simultaneously injected intraperitoneally to increase the volume and protein concentration of the saliva, respectively. The protein composition and relative protein-specific abundance of saliva secreted by the parotid gland and by the mandibular and monostomatic sublingual gland, were determined using iTRAQ. When combining two detection methods, MALDI-TOF/TOF MS and Q-Exactive orbitrap MS/MS, a total of 122 porcine salivary proteins and 6 mammalian salivary proteins with a predicted porcine homolog were identified. Only a quantitative and not a qualitative difference was observed between both ductal secretions. The 128 proteins were detected in both secretions, however, at different levels. Twenty-four proteins (20 porcine and 4 mammalian with a predicted porcine homolog) were predominantly secreted by the parotid gland, such as carbonic anhydrase VI and alpha-amylase. Twenty-nine proteins (all porcine) were predominantly secreted by the mandibular and sublingual glands, for example salivary lipocalin and submaxillary apomucin protein. Data are available via ProteomeXchange with identifier PXD008853. SIGNIFICANCE: In humans, more than 3000 salivary proteins have been identified. To our knowledge, previous studies on porcine saliva only identified a total of 34 proteins. This research increased the total number of identified proteins in porcine saliva to 143. This insight into the porcine salivary proteome will facilitate the search for potential biomarkers that may help in the early detection of pathologies and follow-up of animal welfare. Moreover, it can also endorse the value of a porcine animal model and contribute to a better understanding of the animal's physiology. Additionally, this was the first study to collect and analyse gland specific saliva of pigs. The obtained relative-quantitative knowledge of the identified proteins is valuable when comparing data of stimulated (chewing on a device) vs. unstimulated (passive) saliva collection in the future, since salivary stimulation changes the relative contribution of the major salivary glands to the whole saliva in the oral cavity. For example, carbonic anhydrase VI, which is present in higher concentrations in parotid saliva, has a higher concentration in stimulated whole saliva because of the larger contribution of the parotid gland after stimulation by chewing.

Saliva Proteomics Analysis Offers Insights on Type 1 Diabetes Pathology in a Pediatric Population.

The composition of the salivary proteome is affected by pathological conditions. We analyzed by high resolution mass spectrometry approaches saliva samples collected from children and adolescents with type 1 diabetes and healthy controls. The list of more than 2000 high confidence protein identifications constitutes a comprehensive characterization of the salivary proteome. Patients with good glycemic regulation and healthy individuals have comparable proteomic profiles. In contrast, a significant number of differentially expressed proteins were identified in the saliva of patients with poor glycemic regulation compared to patients with good glycemic control and healthy children. These proteins are involved in biological processes relevant to diabetic pathology such as endothelial damage and inflammation. Moreover, a putative preventive therapeutic approach was identified based on bioinformatic analysis of the deregulated salivary proteins. Thus, thorough characterization of saliva proteins in diabetic pediatric patients established a connection between molecular changes and disease pathology. This proteomic and bioinformatic approach highlights the potential of salivary diagnostics in diabetes pathology and opens the way for preventive treatment of the disease.

Comparative proteomic analysis of the influence of gender and acid stimulation on normal human saliva using LC/MS/MS.

PURPOSE: Human saliva is an important source for disease biomarker discovery. This study is to investigate the influence of gender and acid stimulation on the normal human salivary proteome. EXPERIMENTAL DESIGN: Unstimulated and acid-stimulated saliva samples from 5 males and 5 females were labeled with 4-plex iTRAQ and analyzed by 2-DLC MS/MS. By bioinformatics analysis the gender and acid stimulation related proteins were defined. According to protein annotation the important proteins were validated by multiple reaction monitor analysis. RESULTS: A total of 1770 proteins were identified, and 82 proteins in unstimulated saliva were found to be gender-specific, mainly associated with immune function, metabolism and inflammation. However, no gender-specific proteins were found in acid-stimulated saliva. In addition, 182 and 307 differential proteins were found to be acid stimulation-specific in male samples and female samples, respectively, mainly participated in the process of cellular movement, immune function and inflammatory response. Besides, it was found that acid stimulation caused more significant alteration and played a more important role in the human salivary proteome than gender. Gender-specific (IGHG2 and TIMP1) and acid stimulation (PERL, ENOA, ACTB, B4E022 and CALL3) related proteins were validated by MRM analysis. CONCLUSIONS AND CLINICAL RELEVANCE: The results indicate that gender differences exist in the unstimulated salivary proteome, and the influence of acid stimulation on the salivary proteome was more significant than that of gender. The above results may be helpful for salivary proteome research in the future.