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People with Down syndrome, a frequent genetic disorder in humans, have increased risk of health problems associated with this condition. One clinical feature of Down syndrome is the increased prevalence and severity of periodontal disease in comparison with the general population. Because saliva plays an important role in maintaining oral health, in the present study the salivary proteome of Down syndrome subjects was investigated to explore modifications with respect to healthy subjects. Whole saliva of 36 Down syndrome subjects, divided in the age groups 10-17 yr and 18-50 yr, was analyzed by a top-down proteomic approach, based on the high performance liquid chromatography-electrospray ionization-MS analysis of the intact proteins and peptides, and the qualitative and quantitative profiles were compared with sex- and age-matched control groups. The results showed the following interesting features: 1) as opposed to controls, in Down syndrome subjects the concentration of the major salivary proteins of gland origin did not increase with age; as a consequence concentration of acidic proline rich proteins and S cystatins were found significantly reduced in older Down syndrome subjects with respect to matched controls; 2) levels of the antimicrobial α-defensins 1 and 2 and histatins 3 and 5 were significantly increased in whole saliva of older Down syndrome subjects with respect to controls; 3) S100A7, S100A8, and S100A12 levels were significantly increased in whole saliva of Down syndrome subjects in comparison with controls. The increased level of S100A7 and S100A12 may be of particular interest as a biomarker of early onset Alzheimer's disease, which is frequently associated with Down syndrome.

Proteomic platforms can be classified in bottom-up strategies, which analyze the sample after proteolytic digestion, and top-down strategies, which analyze the intact naturally occurring proteome. Bottom-up platforms are high-throughput because they can investigate a large number of proteins, regardless of their dimension. Nonetheless, information on post-translational modifications (PTMs) can be lost, especially those regarding naturally occurring cleavages and alternative splicing. Top-down platforms cannot cover vast proteomes, however, they can disclose subtle structural variations occurring during protein maturation and allow label-free relative quantifications in an unlimited number of samples. A repertoire of 256 masses belonging to naturally occurring proteins and peptides consistently detected by RP-HPLC-ESI-MS analysis of the acidic soluble fraction of human whole saliva is presented in this study. Of them, 233 have been identified, while 23 are still pending for the definitive characterization. The present review reports average and mono-isotopic masses of the peptides and proteins detected, RP-HPLC elution times, PTMs, origin and quali-quantitative variations observed in several physiological and pathological conditions. The information reported can be a reference for users of top-down RP-HPLC-ESI-MS proteomic platforms applied to the study of the human salivary proteome as well as of other human bodily fluids.

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 β(4) and β(10), antileukoproteinase, histone H1c, and α and γ 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.

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.

To elucidate the localization of post-translational modifications of different classes of human salivary proteins and peptides (acidic and basic proline-rich proteins (PRPs), Histatins, Statherin, P-B peptide, and "S type" Cystatins) a comparative reversed phase HPLC-ESI-MS analysis on intact proteins of enriched granule preparations from parotid and submandibular glands as well as parotid, submandibular/sublingual (Sm/Sl), and whole saliva was performed. The main results of this study indicate the following. (i) Phosphorylation of all salivary peptides, sulfation of Histatin 1, proteolytic cleavages of acidic and precursor basic PRPs occur before granule storage. (ii) In agreement with previous studies, basic PRPs are secreted by the parotid gland only, whereas all isoforms of acidic PRPs (aPRPs) are secreted by both parotid and Sm/Sl glands. (iii) Phosphorylation levels of aPRPs, Histatin 1, and Statherin are higher in the parotid gland, whereas the extent of cleavage of aPRP is higher in Sm/Sl glands. (iv) O-Sulfation of tyrosines of Histatin 1 is a post-translational modification specific for the submandibular gland. (v) The concentration of Histatin 3, Histatin 5, and Histatin 6, but not Histatin 1, is higher in parotid saliva. (vi) Histatin 3 is submitted to the first proteolytic cleavage (generating Histatins 6 and 5) during granule maturation, and it occurs to the same relative extent in both glands. (vii) The proteolytic cleavages of Histatin 5 and 6, generating a cascade of Histatin 3 fragments, take place after granule secretion and are more extensive in parotid secretion. (viii) Basic PRPs are cleaved in the oral cavity by unknown peptidases, generating various small proline-rich peptides. (ix) C-terminal removal from Statherin is more extensive in parotid saliva. (x) P-B peptide is secreted by both glands, and its relative quantity is higher in submandibular/sublingual secretion. (xi) In agreement with previous studies, S type Cystatins are mainly the product of Sm/Sl glands.

In the present study the analytical potential of HPLC-MS/MS was utilized for the structural characterization of a post-translational modification of statherin. Human salivary statherin (M(av)5380.0 +/- 0.3 Da) is transformed by the action of transglutaminase 2 into a cyclic derivative with an average molecular mass of 5363.0 +/- 0.3 Da. The intra-molecular bridge is generated by the loss of an ammonia molecule between the unique Ione-pair donating nucleophile Lys-6 and one acceptor among the seven glutamine residues of statherin. Digestion of the cyclic derivative with chymotrypsin, proteinase K, and carboxypeptidase Y, monitored by HPLC-electrospray ionization-ion trap-mass spectrometric analysis, demonstrated that cyclization involved almost specifically Gln-37 (> 95%), with the percentage of Gln-39 implicated in the cross-linkiing being less than 5%. The main derivative was named cyclostatherin Q37. Guineapig transglutaminase 2 showed high affinity for statherin in vitro (Km = 0.65 +/- 0.06 microM). Cyclo-statherin was detected in vivo by HPLC-electrospray ionization ion trap-mass spectrometry analysis of whole human saliva and it accounted for about 1% of total statherin. Detection of cyclo-statherin in whole saliva is suggestive of a putative role of this molecule in the formation of the "oral protein pellicle".

The acidic-soluble protein content of human gingival crevicular fluid was analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC), and the eluent deriving from the chromatography separation was directly introduced into an ion-trap mass spectrometer through electrospray ionization (ESI-IT MS). By this technique the molecular weight of peptides/proteins was determined with a precision of approximately 1/10,000 amu. On the basis of the chromatographic behavior and the knowledge of the molecular mass value, some peptides and proteins soluble in acidic solution were unambiguously recognized. Besides high quantities of human serum albumin, alpha-defensins 1-4 and minor amounts of cystatin A, statherin, basic PB salivary peptide and other unidentified components were detected. The presence of alpha-defensins in gingival crevicular fluid is in agreement with their relevant contribution to protein composition deriving from granulocyte secretions. Other peptides and proteins abundant in human saliva, such as proline-rich proteins (PRPs) and histatins, were not detected in gingival crevicular fluid. Further investigations will be necessary to establish the origin of statherin and PB salivary peptide in gingival crevicular fluid.