The Surprising Composition of the Salivary Proteome of Preterm Human Newborn

Abstract

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...

Significant Modifications of the Salivary Proteome Potentially Associated with Complications of Down Syndrome Revealed by Top-down Proteomics

“…Characterization of Salivary Peptides and Proteins-Proteins (salivary acidic proline-rich proteins, histatins, salivary cystatins, statherin, proline-rich peptide P-B, ␣-defensins 1-4, cystatins A, B, C, beta-thymosins (4 and 10), S100A7 (D27), S100A8, S100A9, and S100A12 proteins) and several derivatives (acetylated, cysteinylated, glutathionylated, phosphorylated, and oxidized forms) have been already identified in our previous studies (13)(14)(15)(16)(17)(18)(19)(20)(21). Experimental mass values were obtained by deconvolution of averaged ESI-MS spectra automatically performed using MagTran 1.0 software (22).…”

“…1. Characterization of these proteins, mainly based on the RP-HPLC-ESI-MS and Tandem/MS analysis, has been previously described by some of us (13)(14)(15)(16)(17)(18)(19)(20)(21). Cystatin C and its sulfoxide derivative on methionine 14 were confirmed in the present study by high resolution HPLC-ESI-MS/MS analysis of trypsin digestion products and data are reported in supplemental Table S1.…”

“…Previously, on whole saliva of newborns the isoform E27 was also detected, but in minor quantities with respect to the D27 isoform (20). Protein sequence polymorphisms arising from single nucleotide polymorphisms has been also shown for other human salivary proteins, i.e.…”

Association of high levels of α-defensins and S100A proteins with Candida mannan detection in bronchoalveolar lavage fluid of preterm neonates

“…After collection, specimens were centrifuged at 1,000g for 3 min. Cells were suspended in 250 µl of 0.9% sodium chloride, and the absolute and differential cell counts were obtained by automatic analyzer (Siemens-ADVIA 120; Hematology System, Tarrytown, NY) and by spinning 25 µl of suspended cells onto two glass slides using May-Grunwald-Giemsa stain, as previously described (38). Trifluoroacetic acid 0.2% (vol/ vol) solution was immediately added to cell-free supernatants of BALF samples in 1:1 (vol/vol) ratio, and the solution was centrifuged at 8,000g for 5 min.…”

Safety, dosing, and pharmaceutical quality for studies that evaluate medicinal products (including biological products) in neonates

“…Urine and saliva may be collected noninvasively, but the interpretation of medicinal product analyses can be challenging. The use of salivary diagnostics and genomics has grown in importance and accuracy in correlating with blood levels of various proteins and genes (85,86). Within the next several years, saliva may be used routinely for PK or other laboratory studies if issues relating to transport into saliva and binding proteins can be resolved.…”