Saliva contains a complex mixture of proteins and peptides as well as fragments derived from these molecules. By RP 1 -HPLC-ESI-MS analysis of the acidic soluble fraction of human whole saliva we have identified in the chromatographic pattern more than 120 different proteins and naturally occurring peptides (1-6). Their characterization was performed by a variety of mass spectrometric techniques coupled with different enzymatic treatments and amino acid sequencing. The proteins and naturally occurring peptides belong to families of well characterized salivary proteins including Histatins, Statherin, acidic and basic proline-rich proteins (aPRP and bPRP), Cystatins, and Defensins (1-6). Two-dimensional gel electrophoresis has also been used by other researchers for analysis of salivary proteins and peptides, but this technique is not well suited for identification of small peptides as illustrated by the difficulty in identifying Histatins and the majority of bPRPs and bPRP fragments (7-9). However, knowledge of salivary proteins and peptides as well as their naturally occurFrom the ‡Dipartimento di Scienze Applicate ai Biosistemi, Università di Cagliari,
Saliva is a body fluid of a unique composition devoted to protect the mouth cavity and the digestive tract. Our high performance liquid chromatography (HPLC)-electrospray ionization-MS analysis of the acidic soluble fraction of saliva from preterm human newborn surprisingly revealed more than 40 protein masses often undetected in adult saliva. We were able to identify the following proteins: stefin A and stefin B, S100A7 (two isoforms), S100A8, S100A9 (four isoforms), S100A11, S100A12, small proline-rich protein 3 (two isoforms), lysozyme C, thymosins  4 and  10 , antileukoproteinase, histone H1c, and ␣ and ␥ globins. The average mass value reported in international data banks was often incongruent with our experimental results mostly because of post-translational modifications of the proteins, e.g. acetylation of the N-terminal residue. A quantitative label-free MS analysis showed protein levels altered in relation to the postconceptional age and suggested coordinate and hierarchical functions for these proteins during development. In summary, this study shows for the first time that analysis of these proteins in saliva of preterm newborns might represent a noninvasive way to obtain precious information of the molecular mechanisms of development of human fetal oral structures. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.003467, 1-14, 2011.Saliva is a body fluid of a very complex and specific composition devoted to the protection and well-being of the oral cavity and, because it is swallowed, of the digestive tract (1). Protection is ensured by organic and inorganic solutes and specific peptides and proteins, such as acidic and basic proline-rich proteins, ␣-amylases, salivary cystatins, histatins, and statherin (2-5). In a previous study (6), we have established that some salivary proteins and peptides reach the levels typically observed in the adult around 18 years of age. Encouraged by the noninvasive specimen collection, we explored the salivary protein composition of at-term and preterm newborns, in order to establish the starting point of the secretion of the proteins and peptides specific of saliva. Our first study (7) showed that acidic proline-rich proteins secretion started, although at very low levels, at 7 months of postconceptional age. At this age the level of phosphorylation of these proteins was low and it increased reaching a value comparable with that of adults at about one year of age, in concomitance with the beginning of deciduous dentition. Other deep differences between human and preterm saliva were however evident. Highly abundant protein masses detected in preterm saliva were undetectable (at the sensitivity level of our MS apparatus) or at very low level in adult saliva. In a previous study (8) we identified, by different MS approaches, thymosin  4 (T 4 ) and thymosin  10 (T 10 ) in preterm newborn saliva and established by immunohistochemistry their presence in fetal salivary glands. This finding let us to suppose that in preterm newborns these peptides derived from glan...
Physiological variability of the naturally occurring, human salivary secretory peptidome was studied as a function of age. The qualitative and quantitative changes occurring in the secretion of proteins/peptides specific to the oral cavity (i.e., basic salivary proline-rich proteins, salivary acidic proline-rich phosphoproteins, statherin, proline-rich peptide P-B, salivary cystatins, and histatins) were investigated by high-performance liquid chromatography-electrospray ionization-mass spectrometry in 67 subjects aged between 3 and 44 years. Subjects were divided into five age groups: group A, 8 donors, 3-5 years; group B, 11 donors, 6-9 years; group C, 20 donors, 10-12 years; group D, 15 donors, 13-17 years; group E, 13 donors, 24-44 years. Basic salivary proline-rich proteins, almost undetectable in the 3-5 and 6-9 years groups, reached salivary levels comparable to that of adults (24-44 years) around puberty. Levels of peptide P-D, basic peptide P-F, peptide P-H, peptide P-J (a new basic salivary proline-rich protein characterized in this study), and basic proline-rich peptide IB-1 were significantly higher in the 10-12-year-old group than in the 3-5-year-old group, whereas the increase of proline-rich peptide II-2 was significant only after the age of 12 years. The concentration of salivary acidic proline-rich phosphoproteins, histatin-3 1/24, histatin-3 1/25, and monophosphorylated and diphosphorylated cystatin S showed a minimum in the 6-9-year-old group. Finally, the histatin-1 concentration was significantly higher in the youngest subjects (3-5 years) than in the other groups.
Thymosin beta(4) (Tbeta(4)), its sulfoxide, and thymosin beta(10 )(Tbeta(10)) were detected in human saliva and identified by different strategies based on RP HPLC coupled to electrospray multidimensional IT MS. Tbeta(4 )was almost always detected in whole saliva, its sulfoxide sporadically, Tbeta(10) rarely. Tbeta(4) was undetectable in parotid saliva and less concentrated in submandibular/sublingual saliva than in whole saliva. Analysis of gingival crevicular fluid revealed high relative amounts of Tbeta(4), Tbeta(4) sulfoxide, and Tbeta(10) in all the samples. Tbeta(4) mean concentration was 200 times higher in crevicular fluid (20 micromol/L, N = 9) than in whole saliva (0.1 micromol/L, N = 9). Crevicular fluid concentration of Tbeta(4 )(ca. 5% represented by its sulfoxide) and beta(10 )significantly correlated (r = 0.856; N = 9), and their ratio was about 5. A significant correlation was also observed between Tbeta(4 )concentrations in whole saliva and gingival crevicular fluid (r = 0.738; N = 9). Immunohistochemical analysis of the major salivary glands showed that immunoreactivity for Tbeta(4) is restricted to ductal cells, with minor degree of focal positivity in some acinar cells. On the whole, results indicate that gingival sulcus is a main, although not the sole, source for oral Tbeta(4 )and Tbeta(10).
Alzheimer disease (AD) is the most prevalent neurodegenerative disease in the elderly, characterized by accumulation in the brain of misfolded proteins, inflammation, and oxidative damage leading to neuronal cell death. By considering the viewpoint that AD onset and worsening may be influenced by environmental factors causing infection, oxidative stress, and inflammatory reaction, we investigated the changes of the salivary proteome in a population of patients with respect to that in healthy controls (HCs). Indeed, the possible use of saliva as a diagnostic tool has been explored in several oral and systemic diseases. Moreover, the oral cavity continuously established adaptative and protective processes toward exogenous stimuli. In the present study, qualitative/quantitative variations of 56 salivary proteoforms, including post-translationally modified derivatives, have been analyzed by RP-HPLC-ESI-IT-MS and MS/MS analyses, and immunological methods were applied to validate MS results. The salivary protein profile of AD patients was characterized by significantly higher levels of some multifaceted proteins and peptides that were either specific to the oral cavity or also expressed in other body districts: (i) peptides involved in the homeostasis of the oral cavity; (ii) proteins acting as ROS/RNS scavengers and with a neuroprotective role, such as S100A8, S100A9, and their glutathionylated and nitrosylated proteoforms; cystatin B and glutathionylated and dimeric derivatives; (iii) proteins with antimicrobial activity, such as α-defensins, cystatins A and B, histatin 1, statherin, and thymosin β4, this last with a neuroprotective role at the level of microglia. These results suggested that, in response to injured conditions, Alzheimer patients established defensive mechanisms detectable at the oral level. Data are available via ProteomeXchange with identifier PXD021538.
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