6.1. Application of salivary proteomics for diagnostics of oral diseases

The use of proteomics for diagnostics and treatment of oral diseases has been described in a number of publications. Jancsik et al. [70] describe the use of salivary proteomics to identify squamous oral cancer in diabetes patients. Authors have performed an additional sample homogenization, which is rarely described in other approaches to analyze saliva. Following analysis was performed by applying 2D gel separation of proteins and MS analysis using MALDI-TOF without the previous chromatographic separation and fractionation of proteins. It is known that inflammatory processes have a well-documented carcinogenetic role. Patients suffering from type-2 diabetes have also a higher risk of inflammatory diseases in the gastrointestinal tract such as ulcerative colitis or Crohn's disease. These patients have also a higher risk of developing gastrointestinal cancer. It was shown that the incidence of developing benign tumors, leukoplakia, and malignancies was significantly increased in the group of patients with diabetes than in the healthy control group. The authors have shown a discovery of several putative biomarkers such as, e.g. Annexin A8-like, Annexin A8-like 1, Tyrosine kinase, AX969656, Protein kinase, Peroxiredoxin-2, and Annexin A2. Annexins are known to be overexpressed in colorectal cancer but also to have altered in tumorigenesis in several types of tumor. Furthermore, loss of Annexin A1 has been found to be an early event in esophageal squamous cell carcinoma. Obviously, these results show that diabetic patients have a higher risk of developing esophageal squamous cell carcinoma than the control healthy group and close monitoring shall be applied for early detection.

Delaleu et al. [71] have performed a particularly interesting and thorough investigation of the salivary proteome from patients suffering from Sjörgen's syndrome. Salivary proteome was analyzed using a 187-plex capture antibody-based assay, and the salivary proteomic biomarker profiles were generated from patients with primary Sjörgen's syndrome, patients with rheumatoid arthritis, and from asymptomatic controls. Authors were able to characterize putative biomarkers by detecting significant changes in 61 and 55 proteins, respectively, in samples of patients compared to that of donors without the diagnosis of Sjörgen's syndrome. Authors were able to detect, based on 4-plex and 6-plex biomarker signatures, markers

Figure 7. Pathway analysis of salivary proteins using DAVID® resulted in expected output and identification of salivary secretion as the pathway with the highest number of expressed proteins. Data courtesy of Zofia Świątczak (Master Thesis).

76 Salivary Glands - New Approaches in Diagnostics and Treatment

including interleukin-4 (IL-4), IL-5, and clusterin. Accurate prediction of an individual's group membership was achieved for at least 94% of cases.

to proteolysis and evidenced an increased content of some specific protein fragments in saliva, which have been shown to be related with bacterial attachment and the accumulation of phosphopeptides. Especially, the accumulation of phosphopeptides seems to be involved in tooth protection against erosion and the level of their expression and phosphorylation might be a measure for dental damage that can be sustained in diabetes patients. The proteolytic fragments from bPRP1, bPRP2, and aPRP, in particular, might be considered for monitoring the

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Rao et al. [73] applied multidimensional HPLC-MS/MS proteomics analysis for investigation of salivary proteins in patients with Diabetes Mellitus type-2. A Strong Cation Exchange separation of tryptic peptides from saliva of diabetic, prediabetic, and healthy subjects was performed prior to reversed phase separation and MS/MS detection. More than 400 proteins were identified and characterized and label-free quantitation was applied. As with other analysis, proteins involved with metabolic and inflammatory processes were detected in the saliva of subjects with diabetes. An important finding of the study was achieved in the prediabetic saliva: Salivary biomarkers of established Diabetes Mellitus were identified by proteomic profiling to be also differentially abundant in the saliva of patients with impaired glucose tolerance (IGT) alone and IGT + IFG (impaired fructose tolerance). These results were

The authors showed that the relative increase of some of these putative markers is associated with progression of prediabetes to the diabetic state. Therefore, systematic analysis of these putative biomarkers in prediabetic saliva, as well as their variability in individual samples, by immunoassays is of extreme importance for early acting and treatment of patients, which can

An important aspect of patients suffering from DM type-2 is the severe retinopathy that can lead to blindness. Chee et al. [74] performed quantitative proteomics analysis of salivary proteins from patients diagnosed with DM type-2 without retinopathy, which served as controls and patients with DM type-2 and retinopathy. Quantitative proteomics analysis was performed by applying iTRAQ labeling and peptide separation before MS/MS detection was performed on a 50-cm nano column. Authors identified more than 300 proteins but have selected only the fully labeled pairs for quantitative analysis, a total of 119 proteins. Authors identified that increased proteins were predicted to be defense proteins and metabolic proteins suggesting that the expression of salivary defense and metabolic proteins is related to diabetic retinopathy. These results confirmed the report by Fernandez-Real et al. [75] that defense response proteins were elevated in type-2 diabetic patients and this gradually led to surging

6.3. Application of salivary proteomics for psychiatric and neurological diseases

Fields of neurology and psychiatry urgently need new biomarkers for objective and earlier diagnoses of conditions attributed to the central nervous system (CNS). Proteomics and other "omics" technologies are being increasingly applied for these discoveries. Henskens et al. [76] performed analysis of salivary proteins already in 1996 for patients treated with different medications for epilepsy. This work was not performed by using nowadays technology, however, several salivary proteins in saliva from epileptic patients, who were medicated with

disease pathogenesis with potential use for as early detection markers.

further confirmed by direct Western immunoblot and ELISA analyses.

prevent cardiovascular complications and mortality in diabetes patients.

of metabolic proteins.

Winck et al. [72] analyzed the salivary proteome in order to decipher the immune response in oral cancer based on the salivary proteome and the extracellular vesicles isolated from saliva. The authors were able to identify significant differences in processes related to inflammatory and humoral immune responses, to peptidase inhibitor activity, iron coordination, and protease binding. Based on identifications achieved, the two classes of individuals (healthy versus patients with Oral Squamous Cell Carcinoma) were distinguished with 90% accuracy based solely on the proteomics data. The authors have used the label-free approach to quantify the identified proteins. Although both groups of peptides share the great majority of identified proteins, some identified proteins were present only in the healthy or only in the group diagnosed with cancer. Authors described that out of many differentially expressed proteins, only the protein peptidyl-prolyl cis-trans isomerase A (also known as cyclophilin-A) was statistically significant in the analysis of the mean survival time of patients, with reduced abundance of PPIA being a factor that may predict poor prognosis of OSCC patients.

#### 6.2. Application of salivary proteomics for diagnostics of diabetes

Sedentary lifestyle paired with unhealthy food, environmental derogation, and stress situations have led to significant increase in diabetes patients worldwide. Early detection of biomarkers would enable more efficient therapy and possible delay of the diseases onset or even a prevention of the outbreak. Caseiro et al. [67] have described the use of proteomics to study Diabetes mellitus (DM) type-1. Authors have performed a quantitative proteomics analysis using the chemical proteomics approach and chemical labeling using iTRAQ. Here, sample from patients diagnosed with diabetes and from healthy subjects was pooled and processed prior to LC-MS/MS measurement and bioinformatics analysis of generated data. In addition to performing iTRAQ labeling and quantitation, authors have chosen for separating peptides by applying two-dimensional separation using high pH reversed phase chromatography. It is remarkable that authors also identified endogenous salivary peptides that are mostly ignored using MALDI-TOF and combined the results to identify more than 400 proteins. Authors used the data obtained to evaluate protein expression for patients with retinopathy, nephropathy, and no complications with the salivary proteome of healthy donors. Identification of the bactericidal/permeability increasing protein-like 1 (BPI) and pancreatic adenocarcinoma upregulated factor (PAUF) in the saliva of all diabetics clearly suggests that the activation of the immune system in type 1 DM is the most prominent process. One of the proteins, BPI, is an essential component of the innate immune system with bacteriostatic and bactericidal effects against gram-negative bacteria through lipopolysaccharides binding. The PAUF is an endogenous ligand of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4), and it is also involved in the inflammatory response, which seems to be more pronounced and more prominent in patients with retinopathy and nephropathy considering the high salivary levels of alpha-2-macroglobulin, defensin alpha 3 neutrophil-specific, leukocyte elastase inhibitor, matrix metalloproteinase-9, and neutrophil elastase.

As authors also performed analysis of the peptidome, interesting data were obtained that supported the hypothesis that Diabetes Mellitus (DM)-related proteins have higher susceptibility to proteolysis and evidenced an increased content of some specific protein fragments in saliva, which have been shown to be related with bacterial attachment and the accumulation of phosphopeptides. Especially, the accumulation of phosphopeptides seems to be involved in tooth protection against erosion and the level of their expression and phosphorylation might be a measure for dental damage that can be sustained in diabetes patients. The proteolytic fragments from bPRP1, bPRP2, and aPRP, in particular, might be considered for monitoring the disease pathogenesis with potential use for as early detection markers.

including interleukin-4 (IL-4), IL-5, and clusterin. Accurate prediction of an individual's group

Winck et al. [72] analyzed the salivary proteome in order to decipher the immune response in oral cancer based on the salivary proteome and the extracellular vesicles isolated from saliva. The authors were able to identify significant differences in processes related to inflammatory and humoral immune responses, to peptidase inhibitor activity, iron coordination, and protease binding. Based on identifications achieved, the two classes of individuals (healthy versus patients with Oral Squamous Cell Carcinoma) were distinguished with 90% accuracy based solely on the proteomics data. The authors have used the label-free approach to quantify the identified proteins. Although both groups of peptides share the great majority of identified proteins, some identified proteins were present only in the healthy or only in the group diagnosed with cancer. Authors described that out of many differentially expressed proteins, only the protein peptidyl-prolyl cis-trans isomerase A (also known as cyclophilin-A) was statistically significant in the analysis of the mean survival time of patients, with reduced

abundance of PPIA being a factor that may predict poor prognosis of OSCC patients.

Sedentary lifestyle paired with unhealthy food, environmental derogation, and stress situations have led to significant increase in diabetes patients worldwide. Early detection of biomarkers would enable more efficient therapy and possible delay of the diseases onset or even a prevention of the outbreak. Caseiro et al. [67] have described the use of proteomics to study Diabetes mellitus (DM) type-1. Authors have performed a quantitative proteomics analysis using the chemical proteomics approach and chemical labeling using iTRAQ. Here, sample from patients diagnosed with diabetes and from healthy subjects was pooled and processed prior to LC-MS/MS measurement and bioinformatics analysis of generated data. In addition to performing iTRAQ labeling and quantitation, authors have chosen for separating peptides by applying two-dimensional separation using high pH reversed phase chromatography. It is remarkable that authors also identified endogenous salivary peptides that are mostly ignored using MALDI-TOF and combined the results to identify more than 400 proteins. Authors used the data obtained to evaluate protein expression for patients with retinopathy, nephropathy, and no complications with the salivary proteome of healthy donors. Identification of the bactericidal/permeability increasing protein-like 1 (BPI) and pancreatic adenocarcinoma upregulated factor (PAUF) in the saliva of all diabetics clearly suggests that the activation of the immune system in type 1 DM is the most prominent process. One of the proteins, BPI, is an essential component of the innate immune system with bacteriostatic and bactericidal effects against gram-negative bacteria through lipopolysaccharides binding. The PAUF is an endogenous ligand of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4), and it is also involved in the inflammatory response, which seems to be more pronounced and more prominent in patients with retinopathy and nephropathy considering the high salivary levels of alpha-2-macroglobulin, defensin alpha 3 neutrophil-specific, leukocyte elastase inhibitor, matrix

As authors also performed analysis of the peptidome, interesting data were obtained that supported the hypothesis that Diabetes Mellitus (DM)-related proteins have higher susceptibility

6.2. Application of salivary proteomics for diagnostics of diabetes

metalloproteinase-9, and neutrophil elastase.

membership was achieved for at least 94% of cases.

78 Salivary Glands - New Approaches in Diagnostics and Treatment

Rao et al. [73] applied multidimensional HPLC-MS/MS proteomics analysis for investigation of salivary proteins in patients with Diabetes Mellitus type-2. A Strong Cation Exchange separation of tryptic peptides from saliva of diabetic, prediabetic, and healthy subjects was performed prior to reversed phase separation and MS/MS detection. More than 400 proteins were identified and characterized and label-free quantitation was applied. As with other analysis, proteins involved with metabolic and inflammatory processes were detected in the saliva of subjects with diabetes. An important finding of the study was achieved in the prediabetic saliva: Salivary biomarkers of established Diabetes Mellitus were identified by proteomic profiling to be also differentially abundant in the saliva of patients with impaired glucose tolerance (IGT) alone and IGT + IFG (impaired fructose tolerance). These results were further confirmed by direct Western immunoblot and ELISA analyses.

The authors showed that the relative increase of some of these putative markers is associated with progression of prediabetes to the diabetic state. Therefore, systematic analysis of these putative biomarkers in prediabetic saliva, as well as their variability in individual samples, by immunoassays is of extreme importance for early acting and treatment of patients, which can prevent cardiovascular complications and mortality in diabetes patients.

An important aspect of patients suffering from DM type-2 is the severe retinopathy that can lead to blindness. Chee et al. [74] performed quantitative proteomics analysis of salivary proteins from patients diagnosed with DM type-2 without retinopathy, which served as controls and patients with DM type-2 and retinopathy. Quantitative proteomics analysis was performed by applying iTRAQ labeling and peptide separation before MS/MS detection was performed on a 50-cm nano column. Authors identified more than 300 proteins but have selected only the fully labeled pairs for quantitative analysis, a total of 119 proteins. Authors identified that increased proteins were predicted to be defense proteins and metabolic proteins suggesting that the expression of salivary defense and metabolic proteins is related to diabetic retinopathy. These results confirmed the report by Fernandez-Real et al. [75] that defense response proteins were elevated in type-2 diabetic patients and this gradually led to surging of metabolic proteins.

#### 6.3. Application of salivary proteomics for psychiatric and neurological diseases

Fields of neurology and psychiatry urgently need new biomarkers for objective and earlier diagnoses of conditions attributed to the central nervous system (CNS). Proteomics and other "omics" technologies are being increasingly applied for these discoveries. Henskens et al. [76] performed analysis of salivary proteins already in 1996 for patients treated with different medications for epilepsy. This work was not performed by using nowadays technology, however, several salivary proteins in saliva from epileptic patients, who were medicated with different antiepileptic drugs (namely phenytoin, valproate, and carbamazepine), were found to be increased and were compared with protein levels in the saliva of healthy control subjects. It was also found that, for all patient groups, the specific amylase activity was increased up to twofold. On the other side, absolute and relative concentrations of cystatin S were diminished in all samples, but particularly strong in patients using either valproate or phenytoin. These data suggest that use of antiepileptic drugs over long periods may cause a decrease of salivary proteins such as sIgA and cystatins, which are involved in the protection of the oral cavity against microbial infections and, therefore, these patients suffer more complications related to gingiva and oral cavity in general.

three types: type 1 (DS I), type 2 (DS II), and type 3 (DS III), referring to clinically localized mild, localized moderate, and generalized tissue inflammation [80]. Authors have performed a quantitative proteomics analysis based on label-free quantitation and using two different MS platforms—an Orbitrap instrument and a Time-of-flight instrument. Interestingly, proteins were detected as differentially expressed between the two LC/MS systems. Protein expression was also different depending on the severity of DS. Authors have observed different levels of protein expression between different stages of DS and between the number of identified and quantified proteins for different disease stages and have identified serum proteins in the saliva of patients with DS III, e.g. ceruloplasmin, hemoglobins, serotransferrin, and albumin, which suggest that DS III patients experience higher level of inflammation and protein leakage from

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An interesting approach has been undertaken by Kaczor-Urbanowicz et al. [82] to study tooth absorption during an inflammatory process caused by orthodontic tooth movement. The orthodontically induced inflammatory root resorption (OIIRR) occurs as a consequence (the most prevalent and unavoidable) of orthodontic tooth movement. Authors have applied 2Dgel separation for salivary proteins upon depletion of abundant proteins (amylase, serum albumin, and IgG), and separated the tryptic peptides using HPLC followed by MS/MS detection. Identified proteins were quantified using the label-free approach. Authors were able to identify more than 700 proteins, which were revealed by quantitative MS of which different numbers were identified for different groups of patients. The strength of this study lays with the depth of the analyzed proteome and the significance of the results. Authors performed the bioinformatic analysis for proteins, which were found to be more than threefold increased. Different patient groups revealed different results although they all have been diagnosed with the tooth resorption. The moderate-to-severe root resorption young group revealed 38 functional clusters associated with acute and dynamic processes and in the moderate-to-severe root resorption adult group, other 16 functional clusters were found and those were related to less dynamic and slower processes. For the young group, these processes included the regulation of acute inflammatory response, defense response, response to stress, response to wounding or healing as opposed to apoptosis, glycoproteins expression, cell adhesion, signal peptides, etc. in the adult group with moderate-to-severe processes. Finally, a number of new putative biomarkers were identified, and these might be used to produce a clinical test that would serve

along with radiography to perform a fast and more reliable diagnosis.

In summary, salivary proteomics is an upcoming approach for both basic and clinical research with a significant potential for use in fast diagnostic approaches. Not only the analysis of salivary proteins but also the analysis of endogenous peptides in saliva and their posttranslational modifications shall be addressed and targeted. Although a number of studies have been published and more are to come, more research is required to validate the discovered putative

blood into saliva.

7. Conclusion

biomarkers so far.

Ngounou Wetie et al. [77] have investigated the use of salivary proteins as possible markers for early onset of Autism Spectrum Disorders (ASDs). Authors have identified increased levels of apolipoproteins apoA1 and apoA4 and of serum paraoxonase/arylesterase 1 (PON1) in ASD sera compared to healthy controls in blood serum and have tested the hypothesis that levels of these peptides might also be elevated in saliva. Authors found statistically significant differences in expression of a number of salivary proteins such as elevated prolactin-inducible protein, lactotransferrin, Ig kappa chain C region, Ig gamma-1 chain C region, Ig lambda-2 chain C regions, neutrophil elastase, polymeric immunoglobulin receptor and deleted in malignant brain tumors 1. Identifications made support the hypothesis that immune system disturbances may be present in individuals with ASDs.

Castagnola et al. [78, 79] have applied a proteomics approach for studying the naturally occurring peptidome of human saliva in children diagnosed with ASDs. The study revealed that naturally occurring peptides in the saliva of children with ASD can bear multiple phosphorylations. The phosphorylation level of four specific salivary phosphopeptides, identified in this study, statherin, histatin 1, and acidic proline-rich proteins for both entire and truncated isoforms, was found to be significantly lower in autistic patients, with hypophosphorylation of at least one peptide observed in 18 ASD subjects (66%). Authors suggest that different phosphorylation and hypophosphorylation of salivary peptides suggest potential asynchronies in the phosphorylation of other secretory proteins. These proteins could be relevant in the development of central nervous system during embryonic development or in early infancy. Furthermore, obtained results suggest that naturally occurring salivary phosphopeptides might help to detect and discriminate a subgroup of ASD patients.

#### 6.4. Application of salivary proteomics for dentistry

Saliva has a continuous and intensive contact and interaction with human teeth and plays an important role in cleaning the tooth surface and antimicrobial defense. Salivary proteomics and its role in dentistry have been studied in a number of experiments. Some of these have addressed the role of salivary proteins in edentulous patients diagnosed with DM. Byrd et al. and Border et al. [80, 81] have addressed the problem of edentulous patients with Denture stomatitis (DS) and DM type-2.

Denture stomatitis refers to an inflammatory condition of the mucosal tissue underneath the denture, which could lead to severe health problems. Clinical classification of DS distinguishes three types: type 1 (DS I), type 2 (DS II), and type 3 (DS III), referring to clinically localized mild, localized moderate, and generalized tissue inflammation [80]. Authors have performed a quantitative proteomics analysis based on label-free quantitation and using two different MS platforms—an Orbitrap instrument and a Time-of-flight instrument. Interestingly, proteins were detected as differentially expressed between the two LC/MS systems. Protein expression was also different depending on the severity of DS. Authors have observed different levels of protein expression between different stages of DS and between the number of identified and quantified proteins for different disease stages and have identified serum proteins in the saliva of patients with DS III, e.g. ceruloplasmin, hemoglobins, serotransferrin, and albumin, which suggest that DS III patients experience higher level of inflammation and protein leakage from blood into saliva.

An interesting approach has been undertaken by Kaczor-Urbanowicz et al. [82] to study tooth absorption during an inflammatory process caused by orthodontic tooth movement. The orthodontically induced inflammatory root resorption (OIIRR) occurs as a consequence (the most prevalent and unavoidable) of orthodontic tooth movement. Authors have applied 2Dgel separation for salivary proteins upon depletion of abundant proteins (amylase, serum albumin, and IgG), and separated the tryptic peptides using HPLC followed by MS/MS detection. Identified proteins were quantified using the label-free approach. Authors were able to identify more than 700 proteins, which were revealed by quantitative MS of which different numbers were identified for different groups of patients. The strength of this study lays with the depth of the analyzed proteome and the significance of the results. Authors performed the bioinformatic analysis for proteins, which were found to be more than threefold increased. Different patient groups revealed different results although they all have been diagnosed with the tooth resorption. The moderate-to-severe root resorption young group revealed 38 functional clusters associated with acute and dynamic processes and in the moderate-to-severe root resorption adult group, other 16 functional clusters were found and those were related to less dynamic and slower processes. For the young group, these processes included the regulation of acute inflammatory response, defense response, response to stress, response to wounding or healing as opposed to apoptosis, glycoproteins expression, cell adhesion, signal peptides, etc. in the adult group with moderate-to-severe processes. Finally, a number of new putative biomarkers were identified, and these might be used to produce a clinical test that would serve along with radiography to perform a fast and more reliable diagnosis.
