## 6 Salivary Proteomics: Techniques and Applications  
Untargeted salivary proteomics is emerging as a promising approach to identifying proteins involved in pathological processes, including periodontitis. In fact, as for untargeted metabolomics, untargeted proteomics consists of the complete analysis of salivary compounds without preselection, with the difference that it analyzes only salivary proteins. Shotgun proteomics is an untargeted analysis approach to examine the whole proteins contained in the sample analyzed. This method relies on bottom-up proteomics, where proteins are enzymatically digested--usually with trypsin--into peptides before being identified and quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Unlike targeted proteomics, shotgun proteomics allows for the broad detection of thousands of proteins in a single experiment, making it particularly useful for the discovery of new biomarkers involved in specific diseases [18; 88; 89]. Most salivary proteins are secreted by salivary glands, their role is to provide viscoelasticity and to allow the coating of oral surfaces made by saliva. Proteins from blood enter saliva in relatively small amounts as part of a transudate, crossing tight junctions between epithelial cells, which present a diffusion barrier. Mucin glycoproteins (MUC5B and MUC7), statherin, proline-rich proteins, carbonic anhydrase 6, hist statins, secretory component, secretory IgA, IgG, albumin, lysozyme, lactoferrin, matrix metalloproteinase-8, interleukin 8, nerve growth factor, leptin, LL37, and alpha-defensin are the principal proteins and peptides detected in saliva that may serve as biomarkers [90]. Besides the role of protecting oral surfaces and providing lubrication [91], salivary proteins are involved in the following processes: antimicrobial defense, in particular, lysozyme, lactoferrin, and defensins are active against bacteria, viruses and fungi, contributing to the immune defense system; anti-inflammatory process, in fact, hist statins and cystatins suppress the production of pro-inflammatory cytokines and inhibit proteases involved in tissue degradation, reducing inflammation and promoting tissue repair and wound healing; digestion; pH regulation; mineralization; and tooth protection [92; 93]. Considering their involvement in the aforementioned processes, variations in their concentrations may reflect para-physiological or pathological conditions. Changes in the salivary levels of components linked to oral diseases have been detected and have been studied in the last decades. For instance, antimicrobial peptides, including LL37 (cathelicidin), alpha-defensins, and beta-defensins, which originate from neutrophils and epithelial cells, can act as indicators of the innate immune response [94]. Moreover, matrix metalloproteinase-8, an enzyme that breaks down collagen and that is released by neutrophils and fibroblasts, has been found in higher concentrations in the saliva of patients with periodontitis. Elevated levels of matrix metalloproteinase-8 and other proteins in the saliva of these patients could serve as indicators for monitoring disease progression and evaluating responses to treatment [95] (Table 4) (Figure 5).