Advances of Proteomic Sciences in Dentistry

Applications of proteomics tools revolutionized various biomedical disciplines such as genetics, molecular biology, medicine, and dentistry. The aim of this review is to highlight the major milestones in proteomics in dentistry during the last fifteen years. Human oral cavity contains hard and soft tissues and various biofluids including saliva and crevicular fluid. Proteomics has brought revolution in dentistry by helping in the early diagnosis of various diseases identified by the detection of numerous biomarkers present in the oral fluids. This paper covers the role of proteomics tools for the analysis of oral tissues. In addition, dental materials proteomics and their future directions are discussed.


Introduction
Every living thing contains fascinating molecules called proteins [1]. Proteins are building blocks for the living matrix and perform various functions [2]. The human body contains a number of different proteins which are structural, catalytic, regulatory, transport and storage, and transducer proteins. Each of these proteins plays a specific functional role [3]. Proteins belong to biological macromolecules that exist as three-dimensional structures because of the sequences involving the twenty different amino acids [4,5]. These amino acids are linked with each other by peptide bonds. The word "proteome", first coined by Mark Wilkins in 1961, is used to describe a mixture of proteins [6]. All proteomes arise from mRNA and can be used to describe a cell's protein content [7]. In simple terms, proteomics is the study of the distribution and interaction of proteins in time and space in a cell, organisms, or an ecosystem. In recent years, a number of proteomical studies on human body fluid and tissues (diseased and non-diseased) have been carried out by several researchers to analyze the chemistry in order to understand the life processes at the molecular as well as the cellular levels [8,9].
Proteomic tools have the ability to analyze human body samples such as blood, saliva, serums, urine, cervico-vaginal fluid (CVF), sperm cells, gingival crevicular fluids (GCF), microorganisms, and different tissues (enamel, dentine, cementum, pulp, gingiva, bone ligaments, stem cells, and mucosa) in both pathological and normal physiological states [10][11][12]. Quite a few studies have reported the Table 1. Detailed discussion of oral diseases protein analysis using proteomic tools.

Sample Disease Condition Proteomic Tools Identified Markers References
Whole mouth saliva (WS) Oral squamous cell carcinoma Shotgun proteome analysis, Western blotting (WB) and Enzyme Linked Immuno-Sorbent Assay (ELISA) MRP14, M2BP, CD59, catalase, profilin, M2BP, involucrin, histone H1, S100A12, and S100P [17] WS   [39] Recent developments in dental proteomic have helped uncover previously unknown details regarding the unique protein structures and their function for the diagnosis, defense mechanisms, and regeneration of dental tissues, tissue calcification, and repairing of dental tissues [40]. The aim of this paper is to elaborate on the currently available techniques, their reported applications for dental tissues. Furthermore, the current status of dental proteomical analysis and the discovered biomarkers is discussed in detail.

Dental Hard Tissue Proteomics
The tooth is the strongest calcified tissue of the human body due to its special architecture and compositions. It is composed of three distinct mineralized hard tissues: enamel, dentine, and cementum. Enamel is the hardest tissue of the human body and contains 96% minerals, 1% proteins and the remainder being water. The adequate mechanical properties of enamel suit its primary function: mastication of food. Enamel, the only dental hard tissue formed before eruption of teeth, is formed by cells called ameloblasts. Histologically, the inorganic component of enamel is composed of micro-rods and inter-rods of hydroxyapatite (HA) crystals embedded in protein matrix, the organic phase [41]. To date, the major enamel proteins that have been recognized are amelogenin, ameloblastin, enamelin, and tuftelin [42]. Additionally, a total of 42 proteins has been identified during enamel formation (secretory phase and maturation phase) by two dimensional electrophoresis (2-DE) and MS. These proteins include ERp29 which is involved in secretory protein synthesis and calcium binding protein (calbindin) and play a role in tooth maturation [43][44][45][46]. It has been concluded that amelogenin takes part in enamel formation and cementum development by guiding cells. It also regulates initiation and growth of HA crystals during the mineralization front across the carboxyl terminals [47,48]. Very recently, a novel organic protein containing enamel matrix was reported in an adult human tooth with thickness of 100-400 µm which could provide important protein transportation or biochemical linkage between enamel and dentin [49]. Ameloblasts secrete enamel specific extracellular matrix protein called ameloblastin and its expression is also detected during the initial development of craniofacial bones and dental hard tissues of mesenchymal origin [50]. The precise role of ameloblastin is not known but it has been hypothesized that it may control the enamel mineralization process during tooth development alongside growth of enamel mineral crystals [51].
The bulk structure of a tooth is made from dentin which possesses neurogenic and regenerative capabilities. By weight, dentin contains 70% minerals (mainly hydroxyapatite), 20% organic component, and 10% water. In proteomics, dentin has been particularly found useful for the identification of collagenous and non-collagenous proteins [52]. Its formation and biomineralization (dentinogenesis) is dynamically complex. Odontoblasts develop and secrete extracellular matrix followed by mineralization in an organized fashion [53]. Most abundant collagenous proteins present in dentin matrix are collagen (type I, III, V, VI, and XII) providing a three dimensional (3D) template for the mineralization of apatite crystals. Fibronectin and matrix metalloproteinase (MMP) 2, 9, and 20 are associated with predentin collagen fibrils [36]. Park et al. [36] performed Sodium-Dodecyl-Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) followed by an LC-MS/MS method for identifying dentin proteins. The outcome of these experiments revealed the presence of 233 proteins and was confirmed using Western blot technique and immunohistochemical staining. This study was the first to provide dentin protein classification such as: metabolic enzymes, signal transduction, cellular organization, transport, immune response, transcription factor activity, cell growth/maintenance, chaperone/stress response, nucleic acid binding, and unknowns function. Another study reported by Jagr et al. [54], 2-DE and nano-LC-MS/MS was used to identify 289 proteins overall of which 90 had been previously unknown. In this study nine novel proteins were identified and were classified as immunoglobulins which help in the formation of extracellular matrix, formation of the cytoskeleton, cell adhesion molecule activity, cytoskeleton protein binding, immune responses, and peptidase activity. These findings may provide deep insight for the regenerative and rehabilitation of dental tissues. Moreover, only a few studies reported the proteomics analysis of cementum and Numerous studies have been conducted on WMS to evaluate various body physiological and pathological conditions and have proven it as a diagnostic as well as a maintenance test fluid. The WMS was isolated from different diseases such as dental caries, Sjögren's syndrome, diabetic patients, breast cancer patients, squamous cell carcinoma patients, and graft-versus-host disease patients. The WMS has been analyzed successfully by proteomical tools (electrophorically and chromatographically) [59][60][61][62].
Human gingival crevicular fluid (GCF) has been analyzed extensively. GCF has a variable protein composition based on periodontal health and diseases. GCF contains serum transudate (found in gingival sulcus), broken products of host epithelial or connective tissues, subgingival microbial plaque, extracellular proteins, host inflammatory mediators and cells [63]. GCF provides medium for the transportation of bacterial byproducts into the periodontal microenvironment and also helps to drive off host derived products [64]. It has been reported that GCF volume for Numerous studies have been conducted on WMS to evaluate various body physiological and pathological conditions and have proven it as a diagnostic as well as a maintenance test fluid. The WMS was isolated from different diseases such as dental caries, Sjögren's syndrome, diabetic patients, breast cancer patients, squamous cell carcinoma patients, and graft-versus-host disease patients. The WMS has been analyzed successfully by proteomical tools (electrophorically and chromatographically) [59][60][61][62].
Human gingival crevicular fluid (GCF) has been analyzed extensively. GCF has a variable protein composition based on periodontal health and diseases. GCF contains serum transudate (found in gingival sulcus), broken products of host epithelial or connective tissues, subgingival microbial plaque, extracellular proteins, host inflammatory mediators and cells [63]. GCF provides medium for the transportation of bacterial byproducts into the periodontal microenvironment and also helps to drive off host derived products [64]. It has been reported that GCF volume for biochemical and proteomics analysis is limited due to severity of tissue inflammation [65]. Many methods are available for the collection of GCF such as paper strips, capillary tubes, gingival wash, and paper cones [63]. In the last decade researchers have favored using paper strip in their research work due to easy insertion into the gingival crevice up to 1 mm of depth without bleeding from periodontal pockets [35]. After collection of the GCF sample it goes through different steps for proteomics analysis, as illustrated in Figure 2. biochemical and proteomics analysis is limited due to severity of tissue inflammation [65]. Many methods are available for the collection of GCF such as paper strips, capillary tubes, gingival wash, and paper cones [63]. In the last decade researchers have favored using paper strip in their research work due to easy insertion into the gingival crevice up to 1 mm of depth without bleeding from periodontal pockets [35]. After collection of the GCF sample it goes through different steps for proteomics analysis, as illustrated in Figure 2. Variety of proteolytic enzymes are identified in GCF, such as collagenase, elastase, and cathepsin B, D, H, and L [66]. These proteolytic enzymes have been reported as destructors of periodontal tissues and have the capability to degrade type-I collagen and glycoproteins [67]. Table 2 describes detailed profiling of GCF proteins, proteomic tools used, and the number of proteins identified. Most commonly reported identified proteins from GCF are actin, keratins, histones, annexins, proteins S100-A9, apolipoprotein A-1, albumin, salivary gland antimicrobial peptides (histatins, HNP-1, -2 & -3, LL-37, statherin), and cystatin B [68,69]. Some immune related proteins present in GCF such as; Ig γ-1 chain C region, Ig γ-3 chain C region, lactoferroxin-C, leukocyte elastase inhibitor, α 1 antitrypsin, heat shock protein β-1, and coronin-1A [70]. Variety of proteolytic enzymes are identified in GCF, such as collagenase, elastase, and cathepsin B, D, H, and L [66]. These proteolytic enzymes have been reported as destructors of periodontal tissues and have the capability to degrade type-I collagen and glycoproteins [67]. Table 2 describes detailed profiling of GCF proteins, proteomic tools used, and the number of proteins identified. Most commonly reported identified proteins from GCF are actin, keratins, histones, annexins, proteins S100-A9, apolipoprotein A-1, albumin, salivary gland antimicrobial peptides (histatins, HNP-1, -2 & -3, LL-37, statherin), and cystatin B [68,69]. Some immune related proteins present in GCF such as; Ig γ-1 chain C region, Ig γ-3 chain C region, lactoferroxin-C, leukocyte elastase inhibitor, α 1 antitrypsin, heat shock protein β-1, and coronin-1A [70]. A protein based oral biofilm, the acquired enamel pellicle (AEP), is formed on tooth surfaces within seconds after mechanical cleaning of the tooth surfaces [75]. It consists predominantly of proteins secreted from major and minor salivary glands, carbohydrates, ions, exogenous proteins, and lipids [76]. Lee and co-workers investigated AEP layer on enamel and quantified 50 proteins through Liquid Chromatography-Electrospray Ionization-Mass Spectrometry(LC-ESI-MS/MS) [77]. This layer amount is approximately 0.5-1 µg per tooth surface and its formation is crucial and the dynamic process is influenced by many factors such as; circadian cycle, biochemical properties of tooth surfaces, proteolytic capacity of the oral micro environment, and the oral microbiota [78]. The fatty acids (FAs) identified in AEP play an essential role in the pellicle formation, bacterial adhesion and protection against pellicle [79]. In-situ study reveals qualitatively and quantitatively a wide range of FAs (C 12 -C 24 ) through gas-chromatography-electrospray ionization/ mass spectrometry (GC-EI/MS), in this study pellicle were formed in-situ on bovine enamel slabs mounted on upper jaw splints and inserted in the mouth of 10 subjects for 3-240 min. Several methods have been used for the collection of AEP over the last four decades e.g., palatal appliances, chemical solubilization techniques, mechanical techniques and soaked membranes method [80]. All these methods reported different compositions due to different routes of collection. Mayhall et al. [81] remounted freshly extracted discs of teeth crowns in a palatal appliance worn by the subject for 1 h. After AEP formation on the specimens, they dipped the appliances in 2% HCl and detected glutamic acid, serine, and glycine but a low amount of proline [81]. In another in vivo study on AEP composition it revealed a high level of glutamic acid and alanine but a significant amount of hexosamines. This study also determined that a different approach of AEP collection varies the composition of AEP [82]. AEP has many function in the oral cavity such as lubrication, regulation of mineral homeostasis, providing defense against microbes and microbial colonization through specific receptors. Siqueira et al. [75] identified 100 plus proteins and peptides from in vivo AEP, and suggested that all play an active role in maintaining oral health. Similarly, histatin peptide has shown protective mechanism against demineralization of the tooth [83]. A total of 130 proteins were identified from AEP using LC-ESI-MS/MS with high confidence which allowed the classification of AEP proteins according to nature of origin, chemical properties, and biological function as shown in Figure 3 [39]. Very recently, another group of researchers has identified 76 proteins from in vivo AEP present on deciduous teeth through mass spectrometry which opens up a diagnostic frontier in pediatric dentistry [84]. A protein based oral biofilm, the acquired enamel pellicle (AEP), is formed on tooth surfaces within seconds after mechanical cleaning of the tooth surfaces [75]. It consists predominantly of proteins secreted from major and minor salivary glands, carbohydrates, ions, exogenous proteins, and lipids [76]. Lee and co-workers investigated AEP layer on enamel and quantified 50 proteins through Liquid Chromatography-Electrospray Ionization-Mass Spectrometry(LC-ESI-MS/MS) [77]. This layer amount is approximately 0.5-1 µg per tooth surface and its formation is crucial and the dynamic process is influenced by many factors such as; circadian cycle, biochemical properties of tooth surfaces, proteolytic capacity of the oral micro environment, and the oral microbiota [78]. The fatty acids (FAs) identified in AEP play an essential role in the pellicle formation, bacterial adhesion and protection against pellicle [79]. In-situ study reveals qualitatively and quantitatively a wide range of FAs (C12-C24) through gas-chromatography-electrospray ionization/ mass spectrometry (GC-EI/MS), in this study pellicle were formed in-situ on bovine enamel slabs mounted on upper jaw splints and inserted in the mouth of 10 subjects for 3-240 min. Several methods have been used for the collection of AEP over the last four decades e.g., palatal appliances, chemical solubilization techniques, mechanical techniques and soaked membranes method [80]. All these methods reported different compositions due to different routes of collection. Mayhall et al. [81] remounted freshly extracted discs of teeth crowns in a palatal appliance worn by the subject for 1 h. After AEP formation on the specimens, they dipped the appliances in 2% HCl and detected glutamic acid, serine, and glycine but a low amount of proline [81]. In another in vivo study on AEP composition it revealed a high level of glutamic acid and alanine but a significant amount of hexosamines. This study also determined that a different approach of AEP collection varies the composition of AEP [82]. AEP has many function in the oral cavity such as lubrication, regulation of mineral homeostasis, providing defense against microbes and microbial colonization through specific receptors. Siqueira et al. [75] identified 100 plus proteins and peptides from in vivo AEP, and suggested that all play an active role in maintaining oral health. Similarly, histatin peptide has shown protective mechanism against demineralization of the tooth [83]. A total of 130 proteins were identified from AEP using LC-ESI-MS/MS with high confidence which allowed the classification of AEP proteins according to nature of origin, chemical properties, and biological function as shown in Figure 3 [39]. Very recently, another group of researchers has identified 76 proteins from in vivo AEP present on deciduous teeth through mass spectrometry which opens up a diagnostic frontier in pediatric dentistry [84].

Dental Soft Tissue Proteomics
This dental pulp is a soft connective tissue that is composed of cells (mesenchymal, odontoblasts, fibroblasts) neural fibers, blood vessels, and lymphatics [85]. Tooth development, nourishment, sensitivity, defense reactions, repair, and regeneration are the main functions of dental pulp [86]. Its unique composition helps in nutrition as well as sensation for external stimuli [87]. Robertson et al. [88] investigated the calcification response from dental pulp against various external stimuli including dental trauma, caries, abrasion or attrition, and tooth retransplantation. Similarly, Yamazoe et al. [89] harvested dental pulp cells in subcutaneous tissues and analyzed its calcified tissues through proteomics. The reason behind this is that stem cells have the potential to form deciduous or permanent pulp cells. In the last decade, dental pulp stem cells have proven value in repairing dentin-pulp complex [90]. Dental pulp contains unique tissue specific proteins and small leucine-rich proteoglycans (biglycan, lumican, and mimecan) [91]. Pääkkönen et al. [92] analyzed gene and protein expression in healthy and carious dental pulp organs for the first time using cDNA microarray and 2D-gel electrophoresis. In their study, slight expression changes were reported due to the high amount of healthy pulp in both conditions. In addition, a total of 96 proteins were identified through 2-DE gel followed by MS/MS techniques. In the same experiment, cDNA microarrays explored the difference of gene expression in carious tissues and no gene differences were detected in 96 detected proteins. In another in vitro study on proteome mapping of odontoblasts-like dental pulp revealed 23 total proteins by 2-DE gel followed by MS [93]. These proteins are comprised of various types of peptides such as cell membrane bound molecules, cytoskeleton, and nuclear proteins and are involved in matrix synthesis and enzyme metabolism. The expression of various recognized proteins (annexin VI, heteronuclear ribonuclear proteins C, collagen type VI, matrilin-2) were confirmed using western blotting (WB) technique and real time-polymerase chain reaction (RT-PCR) analysis. The RNA amplification technique was successfully used to analyze gene expression and protein encoding linked to physiology of dental pulp. Microarray analysis disclosed a total of 362 genes related to pulp expression specifically hence, further classified as protoncogenes, tooth morphogenesis, genes of collagen, DNAse, metallopeptidases, and growth factors [94]. McLachlan et al. [95] studied dental pulp tissues for detailed characterization and molecular changes due to dental caries. A total of 445 genes were identified with two fold or greater difference in the expression level. At least 85 genes were reported abundant in health and 360 more abundant in disease suggesting that this approach may contribute to improved future diagnosis and treatment. Another comprehensive study on human tooth pulp was done by 2D-gel electrophoresis followed by nano-liquid chromatography tandem mass spectrometry (LC/MS). This approach detected 342 proteins in total with a high confidence, and two proteins were distinguished in human samples [37]. Very recently, Eckhard et al. [96] attempted in depth dental pulp proteome with N-Terminome by the help of the terminal amine isotropic labelling of substrates (TAILS) approach and identified 17 missing protein candidates for the Chromosome-centric Human Proteome Project (C-HPP; www.c-hpp.og). Missing proteins can be defined as proteins that show only transcriptomic evidences and an expected sequence (or suggested by homology) or partly detected proteins. Furthermore, there are transcript evidences for the survival of the corresponding proteins available without conclusive mass spectrometry data [97].
Periodontal ligament (PDL) is another fibrous connective tissue containing heterogeneous cell population and type 1collagen fibers abundantly. They play a key role in maintaining PDL space, homeostasis, and anchorage, as well as maintaining and providing regeneration or repair of periodontium in response to disease and mechanical trauma. Only a few studies reported on PDL cellular components at genomics and proteomics level but it is very essential to understand the unique features and functions. Reichenberg et al. [38] reported a first study on periodontal ligament (PDL) fibroblast proteome for understanding physiology and regulation of PDL and identifying disease related protein markers. In this study 900 spots were detected and 117 proteins spot identified with 74 different genes. In another study on exploring the early osteogenic differential protein-profile in human PDL cells [98], 29 differentially expressed proteins during osteogenic differentiations were reported [98] which have been primarily linked to the cell membrane-binding, cytoskeleton, nuclear regulations, matrix synthesis signal conduction and metabolic enzymes [99]. Proteomics may shed light on these complex functional details of these intra-and inter-cellular processes.

Dental Materials Proteomics
Concurrent application of genomics and proteomics have revolutionized dentistry by allowing the identification and characterization of oral tissues (soft, hard, and liquid), and also help in understanding them on the molecular level [10]. By definition, dental materials are those materials or devices which interact with the oral environment in physio-chemical, mechanical, and biological aspects [100][101][102][103][104][105][106]. Hence, dental materials should be biocompatible and interact without causing any toxicity. Many approaches have been used previously to analyze the success of dental materials and failure at a cellular level. Very recently, Ryta et al. [107] studied elution of unreacted triethylene glycol dimethacrylate (TEGDMA) from Smart Dentine Replacement (SDR™), Dentsply International, UK bulk-fill dental composite by using HPLC. In this study, polymerized specimens were treated with four solutions (100% ethanol, 75% ethanol, distilled water, and 100% methanol) with different concentration to evaluate direct dental pulp toxicity of unreacted TEGDMA monomer. It was confirmed through HPLC that the toxicity of unreacted TEGDMA towards dental pulp established during the first hour after the placement of resin. Dental adhesive systems were analyzed by a research group for the quantification of monomer elution and carbon-carbon double bonds in dental adhesive system using reverse-phase HPLC, and observed that no correlation exists between the resin dentin bonding of adhesives and the elution of unreacted monomers [108]. However, further proteomic analysis of materials on the molecular level is needed to understand the changes in proteomes of failed or successful implants. Some of the studies reported in the last decade on proteomics of dental materials are listed in Table 3. Emdogain contains a number of low-molecular-weight proteins (mainly amelogenins), associated with cementogenesis and osteogenesis during tooth development [109] Derhami et al.
Proteomic analysis of human skin fibroblasts grown on titanium: Novel approach to study molecular biocompatibility Gain a better understanding of the molecular basis of biocompatibility of human skin fibroblast on titanium [110] Koin et al.

Analysis of the degradation of a model dental composite
Liquid chromatography mass spectrometry (LC-MS) found leaching of intact BisGMA and several degradation products that contained the bisphenol A moiety from the overlayer into distilled water after 2 weeks of aging [111] Jung et al.

Proteomic analysis in cyclosporin A (CsA)-induced overgrowth of human gingival fibroblast (HGF)
The CsA-treated HGF demonstrated that Prx 1 may play a crucial role in the HGF proliferation induced by CsA and proteomic analysis data provide an efficient approach in understanding the mechanisms of HGF proliferation by CsA [112] Taiyoji et al.
Identification of proteinaceous inhibitors of a cysteine proteinase (an Arg-specific gingipain) from Porphyromonas gingivalis in rice grain, using targeted-proteomics approaches These results suggest that these rice proteins may be useful as nutraceutical ingredients for the prevention and management of periodontal diseases [113] Haigh et al.

Alterations in the salivary proteome associated with periodontitis
Results highlight the predominant involvement of S100 proteins in the host response during periodontitis [113]  The adherence of LA11 and 89C strain to the moderately rough surfaces coated with saliva was more than twice that seen on the smooth saliva coated surfaces. This clearly demonstrates that surface topography is, at least to some degree, maintained in the presence of a saliva coating [115] Zhao et al.
Quantitative proteomic analysis of human osteoblast-like MG-63 cells in response to bio-inert implant material titanium and polyetheretherketone (PEEK) Titanium and polyetheretherketone (PEEK) induces similar response in osteoblast proteome and PEEK causing worse proliferation was related to mRNA processing [116]

Conclusions
With the help of "omics" (genomics, transcriptomics, proteomics, metabolomics, and metagenomics) many hidden compositions, behavior and metabolisms of dental tissues and oral fluids have been analyzed in the last fifteen years. These scientific disciplines helped the gathering of valuable information of the human proteome and will complete the Human Proteome Project (HPP) [117]. Proteomics tools have provided remarkable information regarding dental tissues and oral fluids [118]. The overall analysis on proteomics in dentistry shows that more studies directed toward structural formation, diagnosis, and pathogenesis but very limited studies on evaluation of treatment, prevention of diseases, and prognosis of interventions. To sum up, all proteomic tools can help to fill the gaps of the unexplored aspects of oral health and dental sciences.  Table 3. Ihtesham Ur Rehman performed literature search, provide the guidelines in order to prepare the manuscript and finalized the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.