MS characterization of multiple forms of alpha-amylase in human saliva

Alpha-amylase is a major and well-characterized component of human saliva. Recent proteomic studies suggested that this protein could be observed in more than twenty spots on 2-D gels of salivary proteins. The aim of this work was to investigate this unexpected redundancy. 2-D gel electrophoresis was combined with systematic MALDI-TOF MS analysis. More than 140 protein spots identifying the alpha-amylase were shown to constitute a stable but very complex pattern. Careful analysis of mass spectra and simultaneous hierarchical clustering of the observed peptides and of the electrophoretic features of spots allowed one to define three major groups. A main class grouping 90 spots was shown to correspond to full length alpha-amylases that can be assumed to include isoforms and post-translationally modified forms, a subset of this class being demonstrated to be N-glycosylated. A second group included short alpha-amylases that are differently truncated in a non-random manner, very likely in the oral cavity. The last class grouped alpha-amylase forms showing both the N- and C-terminal sequences of the enzyme but displaying a molecular weight that was up to 50% lower than that of the native protein. It is speculated that the last group of alpha-amylase spots could correspond to proteins submitted to internal deletions prior to the secretion.     

Proteome analysis of glandular parotid and submandibular-sublingual saliva in comparison to whole human saliva by two-dimensional gel electrophoresis

The secretions of the salivary parotid and submandibular-sublingual (SMSL) glands constitute the main part of whole human saliva (WS) in which proline-rich proteins (PRPs) and mucins represent dominant groups. Although proteome analysis had been performed on WS, no identification of PRPs or mucins by 2-DE and MS was achieved in WS and no comprehensive analysis of both glandular secretions is available so far. The aim of this study was to compare the protein map of WS to parotid and SMSL secretions for the display of PRPs and mucins. WS and glandular secretions were subjected to 2-DE and spots were analyzed by MALDI-MS. New components identified in WS were cyclophilin-B and prolyl-4-hydroxylase. Also acidic and basic PRPs as well as the proline-rich glycoprotein (PRG) could now be mapped in WS. Acidic PRPs were found equally in parotid and SMSL secretions, whereas basic PRPs and PRG were found primarily in parotid secretion. Salivary mucin MUC7 was identified in SMSL secretion. Thus, the more abundant proteins of WS can be explained mainly by mixed contributions of parotid and SMSL secretions with only few components remaining that may be derived from local sources in the oral cavity.     

Proteomics and its applications for biomarker discovery in human saliva

Human saliva is a biological fluid with enormous diagnostic potential. Because saliva can be non-invasively collected, it provides an attractive alternative for blood, serum or plasma. It has been postulated that the blood concentrations of many components are reflected in saliva. Saliva harbors a wide array of proteins, which can be informative for the detection of diseases. Profiling the proteins in saliva over the course of disease progression could reveal potential biomarkers indicative of different stages of diseases, which may be useful in medical diagnostics. With advanced instrumentation and developed refined analytical techniques, proteomics is widely envisioned as a useful and powerful approach for salivary proteomic biomarker discovery. As proteomic technologies continue to mature, salivary proteomics have great potential for biomarker research and clinical applications. The progress and current status of salivary proteomics and its application in the biomarker discovery of oral and systematic diseases will be reviewed. The scientific and clinical challenges underlying this approach will also be discussed.     

NO synthesis in human saliva

Human saliva contains nitrate that is converted into nitrite by the activity of facultative, anaerobic bacteria of the oral cavity. Nitrite can be reduced to NO in the acidic gastric milieu; some NO may also form in the mouth at acidic pH values. In this paper, we show that bacteria ( S. salivarius , S. mitis and S. bovis ) isolated from saliva, may contribute to NO production in human saliva. NO formation by bacteria occurs at neutral pH values and may contribute to the antibacterial activity of saliva.     

Concentrations of major elements and mercury in unstimulated human saliva

The aim of this study was a preliminary assessment of a possible role of human saliva in the diagnosis of some physiological and pathological changes in oral and body functions. Reliable procedures for collection and analysis of samples were established in order to assess total concentrations of Ca, K, Mg, Na, P, and Hg in whole unstimulated saliva. Possible relationships between element concentrations and sex, age, smoking, illness conditions, or side effects resulting from the use of drugs were investigated. The effects of stimulated or unstimulated collection procedures, dental prosthesis, and amalgam fillings were also evaluated. Total concentrations of major cations and Hg in whole saliva from 33 healthy adults living in the Siena district showed a coefficient of variation ranging from 11% (P) to 53% (Na) and average values were in the same range of those previously reported for unstimulated saliva. Healthy males had significantly higher concentrations of K, Na, P, and Na/K, Na/Ca, Na/Mg, and Na/P values than females. Age, smoking, dental prosthesis, and amalgam fillings had no significant effects on the concentrations of major elements. On the contrary, concentrations of Hg were positively correlated to the number of amalgam fillings and increased at a rate of about 1.9 μg/L for each filling. No correlations were found between Hg concentrations and those of major elements. Comparisons with literature data showed a different composition (particularly for Na and Hg concentrations) between unstimulated and stimulated saliva. Samples from patients affected by idiopathic pulmonary fibrosis had significantly higher concentrations of K and the maximum value was measured in a patient affected by acute pulmonary edema. This increase was likely the result of pharmacological treatments with tricyclic antidepressants and/or saline solutions. Data reported in this study, although preliminary, contribute to the assessment of levels of major cations (some of them very little investigated) and Hg in whole unstimulated human saliva and provides consistent support for further research on the possible use of this easy accessible matrix as a diagnostic tool of body function changes.     

Trace elements in human parotid saliva

Although various proteins and some electrolytes have been measured in human saliva, little systematic data about the major and minor elemental components of this body fluid have been obtained. In order to obtain such data, concentrations of C, Na, P, Cl, K, Ca, Sc, Cr, Fe, Co, Zn, Se, Br, Rb, Sb, I, and Cs in human parotid saliva were measured by instrumental nuclear methods. The data obtained confirmed the relative lack of Zn in saliva of patients with hypogeusia (decreased taste acuity) and suggested that concentrations of Na, Cl, Br, and Ca followed the order: normals > hypogeusia > hyposmia (decreased smell acuity). To compare concentrations of elements in saliva with those in blood and urine, absolute concentrations were normalized to that of Na through the use of a concept called an enrichment factor. On this basis, parotid saliva is relatively depleted in Se, Zn, and Fe and enriched for most other elements relative to blood plasma indicating that the fluid is not simply a transudate of blood plasma. Using this same technique, saliva composition was found more similar to urine than blood plasma, being relatively depleted in Se, Cs, and Co, being enriched in I, Br, and Cr and having about the same relative concentrations of P, Cl, Zn, Fe, Ca, K, and Rb. As the total body concentrations of many of the enriched elements in saliva are extremely small, their enrichment in saliva suggests special roles for these elements in the oral cavity. Because of its accessibility, ease of collection, and interaction with some body constituents, saliva represents a useful, albeit neglected, tool in the diagnosis of some physiological and pathological changes in body function and in understanding important aspects of trace metal metabolism.     

Detection of Hsp60 in saliva and serum from type 2 diabetic and non-diabetic control subjects

There is increasing evidence that mitochondrial dysfunction and oxidative stress may be integral to the pathogenesis of type 2 diabetes mellitus. Heat shock protein (Hsp60) is a mitochondrial stress protein known to be induced under conditions of mitochondrial impairment. Although this intracellular protein is normally found in the mitochondrion, several studies have shown that this protein is also present in systemic circulation. In this study, we report the presence of elevated levels of Hsp60 in both saliva and serum of type 2 diabetic patients compared to non-diabetic controls. Hsp60 was detectable in the saliva of 10% of control and 93% of type 2 diabetic patients. Levels detected were in the range of 3–7 ng/ml in control and 3–75 ng/ml in type 2 diabetic patients. Serum Hsp60 levels in the range of 3–88 ng/ml were detected in 33% of control subjects, and levels in the range of 28–1,043 ng/ml were detected in 100% of type 2 diabetic patients. This is the first reporting of the presence of mitochondrial stress protein in salivary secretions. The serum Hsp60 levels were 16-fold higher compared to those in saliva, and there was a good positive correlation between salivary and serum Hsp60 levels (r = 0.55). While the exact mechanisms responsible for the secretion of Hsp60 into biological fluids such as saliva and blood are not yet known. The presence of this molecular marker of mitochondrial stress in saliva offers a non-invasive route to further investigate the biological functions of extracellular Hsp60 in type 2 diabetes mellitus and other conditions.     

Evaluation of saliva as a source of human DNA for population and association studies

A simple noninvasive procedure for saliva sample collection and DNA extraction was developed. On average, the amount of human DNA (as measured by a TaqMan-based assay) was about 11.4 microg/mL saliva, which is more than can be obtained from other noninvasive samples such as cheek swabs. However, the presence of large amounts of nonhuman DNA (up to 90% of the total extracted DNA) in saliva samples does necessitate DNA quantitation methods that are specific for human DNA. We were able to reliably and accurately type different genetic markers (mDNA sequences, Y-chromosomal single-nucleotide polymorphisms, and autosomal microsatellite loci) from saliva samples stored for up to 30 days at 37 degrees C, making this method well-suited for field conditions and convenient transportation of samples back to the laboratory. Thus, saliva can be considered a reliable source of DNA for a wide variety of genetic studies.     

Human infant saliva peptidome is modified with age and diet transition

In order to describe developmental changes in human salivary peptidome, whole saliva was obtained from 98 infants followed longitudinally at 3 and 6months of age. Data on teeth eruption and diet at the age of 6months were also recorded. Salivary peptide extracts were characterised by label-free MALDI-MS. Peptides differentially expressed between the two ages, and those significantly affected by teeth eruption or introduction of solid foods were identified by MALDI TOF-TOF and LC ESI MS-MS. Out of 81 peaks retained for statistical analysis, 26 were overexpressed at the age of 6months. Exposure to solid foods had a more pronounced effect on profiles (overexpression of nine peaks) than teeth eruption (overexpression of one peak). Differential peaks corresponded to fragments of acidic and basic PRPs, statherin and histatin. Comparison with existing knowledge on adult saliva peptidome revealed that proteolytic processing of salivary proteins is qualitatively quite comparable in infants and in adults. However, age and diet are modulators of salivary peptidome in human infants.     

Enrichment and identification of glycoproteins in human saliva using lectin magnetic bead arrays

Aberrant glycosylation of proteins is a hallmark of tumorigenesis and could provide diagnostic value in cancer detection. Human saliva is an ideal source of glycoproteins due to the relatively high proportion of glycosylated proteins in the salivary proteome. Moreover, saliva collection is noninvasive and technically straightforward, and the sample collection and storage is relatively easy. Although differential glycosylation of proteins can be indicative of disease states, identification of differential glycosylation from clinical samples is not trivial. To facilitate salivary glycoprotein biomarker discovery, we optimized a method for differential glycoprotein enrichment from human saliva based on lectin magnetic bead arrays (saLeMBA). Selected lectins from distinct reactivity groups were used in the saLeMBA platform to enrich salivary glycoproteins from healthy volunteer saliva. The technical reproducibility of saLeMBA was analyzed with liquid chromatography–tandem mass spectrometry (LC–MS/MS) to identify the glycosylated proteins enriched by each lectin. Our saLeMBA platform enabled robust glycoprotein enrichment in a glycoprotein- and lectin-specific manner consistent with known protein-specific glycan profiles. We demonstrated that saLeMBA is a reliable method to enrich and detect glycoproteins present in human saliva.     

Identification of human whole saliva protein components using proteomics

The determination of salivary biomarkers as a means of monitoring general health and for the early diagnosis of disease is of increasing interest in clinical research. Based on the linkage between salivary proteins and systemic diseases, the aim of this work was the identification of saliva proteins using proteomics. Salivary proteins were separated using two-dimensional (2-D) gel electrophoresis over a pH range between 3-10, digested, and then analyzed by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-TOF mass spectrometry (MS) and tandem mass spectrometry (MS/MS). Proteins were identified using automated MS and MS/MS data acquisition. The resulting data were searched against a protein database using an internal Mascot search routine. Ninety spots give identifications with high statistical reliability. Of the identified proteins, 11 were separated and identified in saliva for the first time using proteomics tools. Moreover, three proteins that have not been previously identified in saliva, PLUNC, cystatin A, and cystatin B were identified.     

Analysis of age and gender associated N-glycoproteome in human whole saliva

Background

Glycoproteins comprise a large portion of the salivary proteome and have great potential for biomarker discovery and disease diagnosis. However, the rate of production and the concentration of whole saliva change with age, gender and physiological states of the human body. Therefore, a thorough understanding of the salivary glycoproteome of healthy individuals of different ages and genders is a prerequisite for saliva to have clinical utility.

Methods

Formerly N-linked glycopeptides were isolated from the pooled whole saliva of six age and gender groups by hydrazide chemistry and hydrophilic affinity methods followed by mass spectrometry identification. Selected physiochemical characteristics of salivary glycoproteins were analyzed, and the salivary glycoproteomes of different age and gender groups were compared based on their glycoprotein components and gene ontology.

Results and discussion

Among 85 N-glycoproteins identified in healthy human saliva, the majority were acidic proteins with low molecular weight. The numbers of salivary N-glycoproteins increased with age. Fifteen salivary glycoproteins were identified as potential age- or gender-associated glycoproteins, and many of them have functions related to innate immunity against microorganisms and oral cavity protection. Moreover, many salivary glycoproteins have been previously reported as disease related glycoproteins. This study reveals the important role of salivary glycoproteins in the maintenance of oral health and homeostasis and the great potential of saliva for biomarker discovery and disease diagnosis.     

Homogeneity and stability studies on sodium, calcium, magnesium, and manganese in human saliva

This article reports on the homogeneity and stability of saliva samples treated and stored under different conditions. The analytes chosen for this evaluation were Na, Ca, and Mg as minor elements and Mn as the trace element. The results obtained show that when the sample was homogenized in an ultrasonic bath, a minimum volume of 0.25 mL was required to consider the sample homogeneous. The stability of the analytes depended on their concentration. The Ca and Mg content seemed to be stable for at least 1 wk when the samples were stored at room temperature, whereas the Mn content seemed to be unstable for 1 d even when the sample was stored at −20°C.     

Detection of different adenosine triphosphatases in human placental brush border membranes

The microvillous membrane of human placental syncytiotrophoblast cells contains a high ATPase activity. The purpose of this study was to characterize this activity and to investigate the presence of vacuolar type H⁺ ATPase in this membrane. Intact brush border membrane vesicles strongly hydrolyzed ATP, reflecting the presence of ATPase on the external side of the membrane. The ATPase activity was entirely Mg²⁺ dependent and increased with pH. At pH 7.5, Vmax was 31.0 ± 1.7 mol/mg/20 min and Km 0.18 ± 0.03 mm ATP. Hydrolysis of ATP was not influenced by the presence of bicarbonate or alkaline phosphatase inhibitors, but at pH 8 it decreased by half following addition of 100 m dicyclohexylcarbodiimide (DCCD). At pH 7.5, 1 mm N-ethylmaleimide (NEM) depressed this activity by less than 5%. Opening the membrane vesicles with 0.1% desoxycholate (DOC) or Triton-X neither revealed any additional ATPase activity nor altered the low sensitivity to NEM. Treatment of these membranes with 1% cholate decreased the ATPase activity by more than 70% and did not enhance the sensitivity of ATP hydrolysis to NEM. 10^–7 m Bafilomycin, which reduced by 56 ± 9% the ATPase activity in dog kidney brash border membranes treated with 0.1% DOC, had no effect on placental brush border membranes subjected to the same procedure. Finally, neither immunocytochemical staining using monoclonal antibody to the Mr 31000 subunit of V-type H⁺ ATPase, nor electron microscopic examination detected the presence of H⁺ ATPase in placental membranes.In conclusion, the placental brush border membrane is the site of a strong ecto ATPase activity which is partially DCCD sensitive. However, our results did not detect the presence of any vacuolar type H⁺ ATPase activity in these membranes.This study was supported by the MRC grant N MA 9565 and by an extramural grant from Baxter Health Care Corporation. The authors are indepted to Dr. Patrick Vinay for this help in providing Bafilomycin and dog kidney membranes, as well as for his insightful discussion.     

A targeted proteomic strategy for the measurement of oral cancer candidate biomarkers in human saliva

Head and neck cancers, including oral squamous cell carcinoma (OSCC), are the sixth most common malignancy in the world and are characterized by poor prognosis and a low survival rate. Saliva is oral fluid with intimate contact with OSCC. Besides non‐invasive, simple, and rapid to collect, saliva is a potential source of biomarkers. In this study, we build an SRM assay that targets fourteen OSCC candidate biomarker proteins, which were evaluated in a set of clinically‐derived saliva samples. Using Skyline software package, we demonstrated a statistically significant higher abundance of the C1R, LCN2, SLPI, FAM49B, TAGLN2, CFB, C3, C4B, LRG1, SERPINA1 candidate biomarkers in the saliva of OSCC patients. Furthermore, our study also demonstrated that CFB, C3, C4B, SERPINA1 and LRG1 are associated with the risk of developing OSCC. Overall, this study successfully used targeted proteomics to measure in saliva a panel of biomarker candidates for OSCC.     

The epithelial cells and cell fragments in human milk

Summary The cell fragments and epithelial cells in human milk were examined in samples obtained from 30 women: 3 of these provided sequential samples at weekly intervals for 110 days. Membrane-bound cytoplasmic fragments in the sedimentation pellet greatly outnumbered the population of intact cells in all samples. Most of the fragments were derived from secretory cells and contained numerous cisternae of rough endoplasmic reticulum, lipid droplets and Golgi vesicles containing casein micelles. Secretory epithelial cells were present in small numbers in all samples and after the 2nd month of lactation replaced the macrophage as the predominant cell type. Ductal epithelial cells represented less than 1% of the total cell population up to 8 days post-partum, but thereafter they were very rarely found. They occurred in aggregates of 2–4 cells and possessed tight junctions that circumscribed the area of cell-cell contact. All samples of milk contained squamous epithelial cells derived from the galactophores and/or the skin of the nipple. Bacteria were often attached to the surface of the squamous cells. The possible relationship between the presence of secretory epithelial cells in milk and the occurrence of milk proteins in the blood of lactating women is discussed.     

Large-scale phosphoproteome of human whole saliva using disulfide-thiol interchange covalent chromatography and mass spectrometry

To date, only a handful of phosphoproteins with important biological functions have been identified and characterized in oral fluids, and these include some of the abundant protein constituents of saliva. Whole saliva (WS) samples were trypsin digested, followed by chemical derivatization using dithiothreitol (DTT) of the phospho-serine/threonine-containing peptides. The DTT-phosphopeptides were enriched by covalent disulfide-thiol interchange chromatography and analysis by nanoflow liquid chromatography and electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The specificity of DTT chemical derivatization was evaluated separately under different base-catalyzed conditions with NaOH and Ba(OH)₂, blocking cysteine residues by iodoacetamide and enzymatic O-deglycosylation prior to DTT reaction. Further analysis of WS samples that were subjected to either of these conditions provided supporting evidence for phosphoprotein identifications. The combined chemical strategies and mass spectrometric analyses identified 65 phosphoproteins in WS; of these, 28 were based on two or more peptide identification criteria with high confidence and 37 were based on a single phosphopeptide identification. Most of the identified proteins (∼80%) were previously unknown phosphoprotein components. This study represents the first large-scale documentation of phosphoproteins of WS. The origins and identity of WS phosphoproteome suggest significant implications for both basic science and the development of novel biomarkers/diagnostic tools for systemic and oral disease states.     

Relationship of HIV RNA and cytokines in saliva from HIV-infected individuals

We measured levels of six cytokines and human immunodeficiency virus (HIV) RNA in saliva from HIV-seropositive individuals and compared salivary cytokine levels in HIV-seropositives and seronegatives. All of the six tested cytokines were detected in saliva although interleukin-1beta, interferon-gamma and interleukin-10 were detected more frequently (90%, 68% and 61% of samples, respectively) than interleukin-6, tumor necrosis factor-alpha and tumor necrosis factor-alpha receptor II (2-17%). There was no significant association between cytokine levels in saliva and plasma suggesting that cytokines were produced locally. Interferon-gamma levels were significantly higher in saliva from HIV-seropositives when compared to seronegatives while interleukin-10 levels were lower in seropositive saliva. Interleukin-10 levels were higher in individuals with low CD4 counts in the seropositive group. HIV RNA was detected in 29% of saliva samples from seropositives and there was a significant correlation between saliva and plasma HIV RNA levels. However, HIV RNA levels in saliva were not significantly associated with any of the saliva or plasma cytokine levels or with CD4 cell numbers. This study shows no association between inflammatory cytokine levels and HIV levels in saliva and suggests that saliva HIV levels are more influenced by blood HIV RNA levels than oral inflammation.     

Chemical synthesis of phosphoseryl-phosphoserine, a partial analogue of human salivary statherin, a protein inhibitor of calcium phosphate precipitation in human saliva

Human salivary secretions are supersaturated with respect to basic calcium phosphates but spontaneous precipitation of these salts from saliva, or surface-induced precipitation of calcium phosphates onto dental enamel, does not normally occur. This unexpected stability has been attributed to the inhibitory activities of two kinds of salivary phosphoproteins: statherin and the acidic, proline-rich phosphoproteins (PRP). Investigation of the structure-function relationships of statherin, the most potent inhibitor of primary (spontaneous) and secondary (seeded) precipitation of calcium phosphate salts in human saliva has been limited to studies of peptide segments obtained from the native peptide by specific proteolysis. Solid phase peptide synthesis (SPPS) is a useful and potentially more flexible alternative. Phosphoserine residues (positions 2 & 3) play critically important roles in the precipitation-inhibition activities of statherin, but SPP synthesis of these phosphorylated peptides is precluded because of the instability of phosphoserine residues in the presence of HF. Thus, this peptide was synthesized by solution-phase methods. The dipeptide possessed substantial inhibitory activity in assays for inhibition of both primary and secondary precipitation of calcium phosphate salts, but was not as active as either N-terminal tryptic hexapeptide of statherin or intact statherin. Syntheses of other model phosphorylated peptides are underway to expand the structure-function relationships.