2.1 Chemicals and reagents

Protease Inhibitor Cocktail for use with mammalian cell and tissue extracts was obtained from Sigma (St. Louis, USA). DL-Dithiothreitol (DTT), urea, thiourea, iodoacetamide, Tris-HCl, mineral oil, Quick Bradford assay kit, and bovine serum albumin were purchased from BioRad (Hercules, USA). Acetone and calcium chloride were purchased from AppliChem (Darmstadt, Germany), and acetonitrile (ACN), and trifluoroacetic acid (TFA) were from Merck (Darmstadt, Germany). For the protein digestion, a porcine trypsin suitable for protein sequencing was obtained from Promega (Madison, WI, USA). All chemicals and reagents including ethanol, methanol, formic acid (FA) and glycerol were of analytical grade, suitable for the electrophoresis and/or for the mass spectrometry measurements. Digested protein extracts were separated on ImmobilineDryStrips (pH 3-10, 13 cm) purchased from GE Healthcare Life Sciences (Little Chalfont, United Kingdom). All solutions were prepared using ultra-pure water produced by MilliQ Integral 3 water purification system from Merck (Darmstadt, Germany).

2.2 Collection of saliva and sample preparation

WUS was collected from volunteers/individuals. The inclusion criteria involved overall systemic health with no current or recent medications. Smokers or occasional smokers were excluded from the study. The individuals who fit the inclusion criteria were classified into two groups: a group of 19 year old volunteers with DMFT = 0 and a caries experienced group with DMFT > 0 (i.e., 4–9). Volunteers were asked to give up the morning oral hygiene and eating 1 h prior to saliva sampling. To reduce effects of circadian rhythm, saliva samples were collected between 8–10 a.m. Approximately 5 ml of saliva was collected by expectoration into sterile 50 ml polypropylene tubes (BD Falcon, BD Bioscience, New Jersey, USA) placed on ice. After collection, the 1 μl of protease inhibitor cocktail per 1 ml of saliva was added to prevent the sample degradation. In the following step, supernatant containing saliva proteins and non-soluble debris, food fragments and bacterial cells were separated by centrifugation at 14000×g for 30 min at 4°C. The pellet was discarded and the supernatant stored at −0°C for further analysis.

Salivary proteins were precipitated by mixing the supernatant with acetone and 0.2% DTT mixed in 1:5 (v/v) ratio. After overnight incubation at −25°C, vortexing followed by centrifugation at 14000×g for 30 min at 4°C was carried out. The pelleted proteins were washed three times with glacial acetone and dried in the vacuum concentrator (CentriVap, Labconco, Kansas City, USA).

2.3 Total protein concentration assay

A Bradford’s assay (Quick Start Bradford Protein Assay, BioRad, Hercules, California, USA) with bovine gamma albumin was used for determining the total level of protein in the solution. Absorption was measured at 595 nm of wavelength using UV-Vis spectrophotometer (UV-3600 Spectrophotometer, Shimadzu corp., Kyoto, Japan).

2.4 In-solution protein digestion

The protein pellet was re-suspended in the 8 mol l⁻¹ urea/100 mmol l⁻¹ Tris-HCl buffer (pH=8) to obtain total protein concentration of 1 mg ml⁻¹. The reduction of disulfide bonds was achieved by addition of 0.1 mol l⁻¹ DTT/100 mmol l⁻¹ Tris-HCl buffer (pH=8) and incubation at 37°C for 30 min with vortexing at 750 rpm. After that, the 0.5 mol l⁻¹ of iodoacetamide/100 mmol l⁻¹ Tris-HCl buffer (pH=8) serving as alkylating reagent was added. The sample was incubated in the dark at 37°C for another 30 min with vortexing at 750 rpm. Afterwards, the proteins were precipitated by pre-cooled acetone. The sample was incubated for 60 min at -25°C, then vortexed and centrifuged at 4000×g for 50 min at 4°C. After discarding the supernatant, the pellet containing proteins was dried in a vacuum concentrator. The proteins were then resuspended in the 8 mol l⁻¹ urea/100 mmol l⁻¹ Tris-HCl buffer (pH=8) to obtain total protein concentration of 1 mg ml⁻¹. The urea concentration was reduced by diluting with 2 mmol l⁻¹ calcium chloride/10 mmol l⁻¹ Tris-HCl buffer up to 2 mol l⁻¹ final concentration of urea. Standard overnight digestion was carried out by adding 1 μl of trypsin (0.1 μg μl⁻¹) to 10 μg of proteins and incubated overnight at 37°C with vortexing at 750 rpm. Trypsin activity was inhibited by acidification with 20% TFA.

2.5 Sample desalting

In the next step, removal of salts, ampholytes and other possibly interfering substances was carried out using solid phase extraction (SPE) cartridges – BondElut C₁₈ 50 mg ml⁻¹ (Agilent Technologies, Santa Clara, CA, USA). Initially, the C₁₈ column was activated by double washing with both the 1 ml ACN:H₂O (50:50, v/v) and the 1 ml H₂O:ACN:TFA (94.5:5:0.5, v/v/v). The 1 ml of sample solution was then loaded onto the SPE column. At first, unbound components were washed off by H₂O:ACN:TFA (94.5:5:0.5, v/v/v). After that, the peptides were eluted from the column with 1 ml ACN:H₂O:FA (70:29.9:0.1, v/v/v) to low adhesion tubes. The sample volume was reduced and the ACN removed by vacuum concentration.

2.6 Off-gel fractionation

In the next stage, the saliva peptides were subjected to electro-migration separation using Agilent 3100 OFFGEL Fractionator (Agilent Technologies, Santa Clara, CA, USA). The sample was diluted with peptide OFFGEL solution containing glycerol and IPG buffer (pH 3-10). The 0.15 ml aliquots were injected per well. A 1 mg of saliva peptides were loaded onto each IPG strip and then separated into 12 fractions in a pH 3-10. The separation was performed according to default standard peptide protocol: OG12PE00. Collected fractions were desalted on SPE cartridges once again and concentrated under vacuum near to dryness.

2.7 Nano-HPLC analysis

Peptides were separated by nano-HPLC system (UltiMate 3000, Dionex, Germany). The trap column (Acclaim PepMap 100, 100 μm×20 mm, 5 μm, 100 Å, Thermo Scientific) and analytical column (Acclaim PepMap RSLC, 75 μm×15 cm, 2 μm, 100 Å, Thermo Scientific) were used as stationary phases for preconcentration and separation, respectively. The mobile phase consisted of 0.1% FA + 98% water + 2% ACN (v/v/v, solution A) and 0.1% FA + 95% ACN + 5% H₂O (v/v/v, solution B) operated at a constant flow rate of 300 nl min⁻¹ during 150 min run-time in the following gradient profile: 10 min at 4% B, 120 min at 4–40% B, 1 min at 40–95% B, 5 min at 95% B, 1 min at 95–4% B and 13 min at 4% B. Fractions of peptides were collected by Proteineer fc II (Bruker Daltonik GmbH, Germany) which collected discrete fractions of the eluted peptides on MALDI target MTP AnchorChip 800/384 TF (Bruker Daltonik GmbH, Germany) every 20 s in the 40–148 min interval. Subsequently, MALDI target was inserted to mass spectrometer for measuring.

2.8 MALDI-TOF/TOF analysis

Mass spectrometry analysis was performed using a MALDI-TOF/TOF UltrafleXtreme (Bruker Daltonik GmbH, Germany). Spectra were acquired in reflectron positive ion mode in the m/z range 700–3500 Da. Alpha-cyano-4-hydroxycinnamic acid (HCCA) mixture was used as the matrix. The 800 μl of the HCCA matrix solution for nano-HPLC fractions was prepared by mixing: 748 μl of TA95 (95% ACN + 5% water solution of 0.1% TFA), 36 μl HCCA saturated in TA90 (90% ACN + 10% water solution of 0.1% TFA), 8 μl of 10% water solution of TFA and 8 μl of 100 mM NH₄H₂PO₄ dissolved in water. The 800 μl of HCCA matrix solution for calibrant spots was prepared using same procedure but TA85 solution (85% ACN + 15% water solution of 0.1% TFA) was used instead of TA95. External mass calibration was performed using the Peptide Calibration Standard II (Bruker Daltonik GmbH, Germany).

2.9 Protein database search

MS a MS/MS spectra were searched by the MASCOT 2.4 search engine (Matrix Science Ltd., UK) against the SwissProt database (December, 2015). Database search parameters were: taxonomy Homo sapiens (human), fixed modification Carbamidomethylation (C), variable modification Oxidation (M), enzyme Trypsin, number of maximum missed cleavages 2, mass error tolerance 100 ppm for MS spectra, 0.5 Da for MS/MS spectra and false discovery rate (FDR) ^< 1.

3.1 Enrichment of the cell component categories assessed by GO

Enrichment maps (Figure 2 A, B) were analyzed by searching for the changes associated with the potential risk factors connected to dental caries. 87 and 83 GO cell component terms were found enriched in the group with DMFT = 0 and DMFT > 0, respectively. 10 GO cell component categories with the smallest p values were selected as significant, depicted in the Table 1 and Table 2.

Figure 2

Cell component networks (GO terms) for proteins in DMFT = 0 (A) and DMFT > 0 (B) group. Color relates to the p-value for the statistical significance of the enrichment of a GO term, while node size is related to the number of proteins associated with a GO term (to distinguish first 10 component categories with smallest p-value apart from others, they were marked with red color).

GO terms in both groups were mainly related to the extracellular region, extracellular space, cytoplasm and cytoskeleton. Membrane-bound vesicles and cytoplasmic membrane-bound vesicle proteins were included in the top ten with the smallest p-values of DMFT = 0, but not DMFT > 0 group, while cytosol and cytoplasmic part GO terms were found significantly enriched in the group with DMFT > 0.

3.2 Enrichment of the molecular function categories assessed by GO

In order to investigate the specific molecular functions represented among the proteins identified in the saliva of caries-free and caries-susceptible individuals, analysis of molecular function annotation was performed. The 84 and the 70 GO molecular function terms were found enriched in the group with DMFT = 0 and DMFT > 0, respectively. Again, 10 GO molecular function categories with the smallest p-values were chosen as significant (Table 3 and Table 4). As shown in Figures 3 and 4, and Tables 3 and 4, protein binding, cytoskeletal protein binding, endopeptidase activity and structural constituent of cytoskeleton categories were ranked as the most significant in both studied groups.

Figure 3

Molecular function networks (GO terms) for proteins of caries-free individuals. Color relates to the p-value for the statistical significance of the enrichment of a GO term, while node size is related to the number of proteins associated with a GO term (to distinguish first 10 component categories with smallest p-value apart from others, they were marked with red color).

Figure 4

Molecular function networks (GO terms) for proteins of caries-susceptible individuals. Color relates to the p-value for the statistical significance of the enrichment of a GO term, while node size is related to the number of proteins associated with a GO term (to distinguish first 10 component categories with smallest p-value apart from others, they were marked with red color).

The rest of the terms of caries-free saliva samples predominantly contained molecular function related to the activity of proteolytic peptidases or its regulation. In contrast to WUS from caries-susceptible individuals, carbohydrate binding was included in the top ten functions of caries-free individuals. In the caries-susceptible group, binding, actin binding, antigen binding and calcium ion binding were included in the top ten with the smallest p-values.

According to Gao et al. [32] salivary proteins are responsible for approximately 50% of the calcium concentration of dental plaque. As such, a change in the protein profile could affect calcium-binding sites. Thus, calcium-binding proteins from saliva can without a doubt serve as a template for mineral growth in dental biofilms, requiring further assessment [33].

Also, searching GO terms for molecular functions in the OralCard web information system (http://bioinformatics.ua.pt/OralCard/diseases/view/68003731) and corresponding proteins associated with dental caries was carried out (Table 5).

To further expand our knowledge about the network associated with caries, the networks were sub-clustered with the aid of the ReactomeFIPlugIn tool and the modules were analyzed. In module 1 of the caries-susceptible group, a branch of cytokine binding genes and gene products not seen in the caries-free network was observed. Interleukin-11 binding, interleukin-11 receptor activity and interleukin-27 receptor activity responsible for the regulation of immune and inflammatory responses to infections were included into this sub-cluster in contrast to caries free-individuals.

In module 2, there were also observed changes that could relate to the mechanism of caries production. Some genes and gene products annotated to hydrolase activity (i.e., phosphoric ester hydrolase activity, bisphosphoglycerate phosphatase activity, 2,3-bisphospho-D-glycerate 2-phosphohydrolase activity) and intramolecule transferase activity (i.e., phosphoglycerate mutase activity, intramolecular transferase activity, phosphotransferases bisphosphoglycerate phosphatase activity, 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase activity, intramolecular transferase activity) were observed only in the oral environment of caries-free individuals.

Water-insoluble glucans produced by cariogenic species S. mutans play an important role in the formation of dental biofilm and adhesion of biofilm to tooth surfaces. Hydrolase-active glucanohydrolases (α-1,3-glucanase, α-1,6-glucanase) are potentially useful for dental caries prevention. Bi-functional chimeric glucanase, composed of α-1,3-glucanase and α-1,6-glucanase, can reduce the formation of the total amount of water-insoluble glucan in a dose-dependent manner, and more effectively decompose biofilm than a mixture of α-1,3-glucanase and α-1,6-glucanase [34].

Transferases belong to enzymes that metabolize sucrose into water insoluble and soluble glucans, which are an integral measure of the biofilm formation initiated by S. mutans. Looking for an ideal treatment preventing dental caries, efforts were taken to explore therapeutic approaches that selectively inhibit the biofilm formation process while preserving the natural bacterial flora of the mouth. Confirming the importance of the presence of transferases in the process of forming tooth decay, it was found that S. mutans glucosyl transferases activity can be inhibited by a group of hydroxychalcones. Leaving commensal and/or beneficial microbes intact, they can serve as potential anti-caries agents [35].

3.3 Enrichment of biological process categories assessed by GO

The hierarchical networks depicted in the Figure 5 and Figure 6 show the variety and interdependence of the biological processes in terms of GO. The 507 and the 397 GO biological process terms were found enriched in the group with DMFT = 0 and DMFT > 0, respectively. The 20 GO biological process categories with the smallest p-values were selected as significant (Table 6 and Table 7). As shown in the Table 6 and Table 7, in the first group these processes associated with the metabolism of carbohydrates dominate. The following processes are not even included in the top 20 for the caries-experienced group: glucose catabolic, monosaccharide metabolic, cellular carbohydrate catabolic, hexose catabolic, monosaccharide catabolic, and glycolysis.

Figure 5

Biological processes networks (GO terms) for proteins in caries-free individuals. Color relates to the p-value for the statistical significance of the enrichment of a GO term, while node size is related to the number of proteins associated with a GO term.

Figure 6

Biological processes networks (GO terms) for proteins caries-susceptible individuals. Color relates to the p-value for the statistical significance of the enrichment of a GO term, while node size is related to the number of proteins associated with a GO term.

Nevertheless, response to chemical stimulus, response to stimulus, immune system process, immune response, defense response, inflammatory response, immune effector process and regulation of immune system process were ranked as the most significant per the results in the group of caries-experienced individuals.

The sub-clusters of networks were selected for detailed analysis. The examination of module 0 also confirmed differences in some branches of the network related to the metabolic processes and their regulation, as well as the regulation of catabolic processes. Figure 7 A, B shows the functional sub-clusters of module 0 representing altered metabolic processes. Table 8 displays a list of GO terms of those processes that occurred in caries-free individuals, but were not recorded in group of caries-susceptible people. However, by searching GO terms for biological processes in the OralCard web information system, only the term glycolysis (P04406 – glyceraldehyde-3-phosphate dehydrogenase) was found as being related to dental caries.

Figure 7

Biological processes (GO terms) of part of sub-networks of module 0 for proteins of caries-free (A) and caries-susceptible (B) individuals. Color relates to the p-value for the statistical significance of the enrichment of a GO term, while node size is related to the number of proteins associated with a GO term. Nodes in white are not significantly overrepresented; they are incorporated to show other nodes in the framework of GO hierarchy.

The influence of other proteins involved in metabolic processes and in the regulation of metabolic and catabolic processes of carbohydrates of caries-free individuals (Table 8) for caries protection was also included.

Module 1 of GO terms of caries-susceptible individuals contained such biological processes as interferon-gamma biosynthetic process, interferon-gamma production, interleukin-13 biosynthetic process, interleukin-13 production, interleukin-2 biosynthetic process, and interleukin-27-mediated signaling pathway, which were not recorded in the caries-free group. Regarding these biological processes, all are linked to the production of cytokines, some of which promote cell-mediated immune functions, organism response to microbial infection, or can induce a class of protein-degrading enzymes known as matrix metalloproteinases [36].

The significantly elevated concentration of the pro-inflammatory cytokines interleukin-6, interleukin-8, and tumor necrosis factor α found in WUS of caries-susceptible subjects, compared to caries-free individuals, confirms their potential impact on the formation of dental caries [37].

Also, the up-regulation of inflammatory cytokines that carry the converged inflammatory signal in the odontoblast layer of human teeth suffering with caries was observed [38]. In another study [39], it was found that cytokines increase defensive capacity, including antimicrobial peptide production, to protect the tooth. Controversially, dental caries was not correlated with salivary or serum concentrations of the studied cytokines of children aged 6-12 years.

3.4 Assessment of proteins using GeneAnalysis tool and OralCard

GeneAnalytics tools (https://ga.genecards.org/#) that provide information about genes of interest as well as precise analysis of how these genes are connected to different diseases and OralCard web information system (http://bioinformatics.ua.pt/OralCard/diseases/view/68003731) were further used to study our experimental data.

Searching GeneAnalytics database revealed 7 proteins encompassed in both groups that are directly associated with dental caries or processes related to them: cystatin SN, lactoperoxidase, lactotransferrin, lysozyme and mucin 7, secreted, are related to dental caries; cathepsin S and glucose-6-phosphate isomerase are related to dentine erosion; glucose-6-phosphate isomerase is related to enamel erosion; glucose-6-phosphate isomerase is related to root caries; and glucose-6-phosphate isomerase is related to tooth erosion.

OralCard database (67 stated proteins) was also used to assess our data and compared well with them (see Table II A in Supplementary material II). The table of caries-experienced individuals included Ig kappa chain V-II region Cum, Ig lambda chain V-I region NEWM, Ig lambda chain V-III region LOI, and keratin, type I cytoskeletal 17, that were not found in the group with DMSF=0, while histone H4 and chitinase-3-like protein 2 were observed only in the group with DMSF>0.

A number of identified salivary proteins that are not listed in GeneAnalytics or OralCard are involved in host defense control in the oral cavity and prevent the growth of microorganisms. Therefore, they could play a role in protecting tooth structure from caries by providing a natural antibiotic barrier. They may also have a function to keep overall bacteria within reasonable limits and help prevent biofilm formation. From our results, the incidence of neutrophil defensin-1 and neutrophil defensin-4 in caries-free subjects is consistent with the study of Hong et al. [18]. They recently confirmed the presence of neutrophil defensin-1 in saliva samples of caries-free subjects but not of caries-positive subjects while studying qualitatively differential profiles of salivary proteins with affinity to S. mutans lipoteichoic acid [18]. Controversially, we identified neutrophil defensin-1 also in the saliva of the caries-susceptible group. Generally, defensins found in neutrophils are cationic antimicrobial peptides, but their role in the protection of dental enamel is still unclear.

Similarly unknown is the role of azurocidin identified in both of our experimental groups. Azurocidin is a neutrophil granule-derived antibacterial and monocyte- and fibroblast-specific chemotactic glycoprotein. There could be a relationship between azurocidin incidence and dental-caries occurrence as it has been detected among unique proteins in caries-free children [40].

Several members of the S100 multigenic calcium-modulated protein family were also detected, as: S100-A2, A7, A8, A9, A11 and A12 (in the caries-free group) and S100-A4, A6, A7, A8, A9, A11 and A12 (in the caries-susceptible group). The S100 protein family consists of 24 members functionally distributed into those which only exert intracellular regulatory effects, those with intracellular and extracellular functions and those which mainly exert extracellular regulatory effects [41]. Their possible relationship to dental-caries was confirmed by immuno-histochemical studies in carious pulpal tissue [42]. PCR analysis in carious and healthy pulpal tissue samples of the S100 family members S100-A8, S100-A9, S100A-12, and S100-A13, indicated that genes tested were more abundantly expressed in carious teeth. The authors showed by gene expression analyses in immune system cells that S100-A8 and the S100-A8/S100-A9 complex were mainly expressed by infiltrating neutrophils and revealed that bacterial activation of neutrophils caused up-regulation of S100-A8, S100-A9, and S100-A13.

Profilin-1 which is classified to the group of proteins responsible for focal adhesion appears also to be of interest from the point of caries development [42]. In another study aimed to compare the proteins of caries-free with caries-susceptible saliva, profilin-1 was identified as a unique protein of caries-free samples [18]. Conversely, we identified profilin-1 in our both experimental groups.

PRPs are divided into the three classes: acidic, basic, and basic glycosylated, representing the most heterogeneous family of human salivary proteins [43]. Whatever the role of PRP species is, it should be noted that PRPs are the most conserved oral salivary proteins among mammals. However, further investigations are needed to clarify their different functions in the oral cavity [44]. The participation of acidic PRPs in the formation of acquired enamel and the development of dental erosion, functioning as predominant pellicle precursor proteins, has been well established. They are involved in the development of the basal layer of the acquired pellicle and control dental erosion by modulating calcium and phosphate concentration within the oral cavity [45]. Moreover, according to Zakhary et al. [46], there is a correlation between acidic PRP alleles of the PRH1 locus (Db) from parotid saliva and caries susceptibility: Db-negative individuals had significantly more caries prevalence.

In our experiment, the proline-rich protein 1, proline-rich protein 4 and three small PRP 2B, 2D and 3 were identified in caries-free subjects while PRP 4 and small PRP 2A, 2B, 2D, 2E, 2F and 3 were observed in caries-susceptible subjects.