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.     

Acute-phase response in dairy cows with acute postpartum metritis

The diagnostic value of 2 plasma acute-phase proteins, haptoglobin and alpha1-acid glycoprotein, and plasma N-acetyl-beta-D-glucosaminidase enzyme activity were studied in 29 newly calved dairy cows. Nineteen had developed acute metritis with putrid vaginal discharge within 2 wk after calving; 10 were clinically healthy controls. Plasma haptoglobin concentration remained low in most cows with acute postpartum metritis. Only the 3 most severely affected cows exhibited a strong haptoglobin response. These were later culled due to poor condition and reduced fertility. This suggests that in acute uterine infection a highly increased haptoglobin concentration indicates poor prognosis for repeat conception. Plasma alpha1-acid glycoprotein concentration increased in acute postpartum metritis, the response pattern being less prominent than that for haptoglobin. The alpha1-acid glycoprotein concentrations did not correlate with severity of disease, and, consequently, the capacity of alpha1-acid glycoprotein in differentiating genital infections was relatively poor. The highest alpha1-acid glycoprotein concentrations were detected in cows with retained placenta and/or dystocia. Plasma N-acetyl-beta-D-glucosaminidase activity levels did not differ between the cows with acute postpartum metritis and healthy control cows.     

Reference maps of mouse serum acute-phase proteins: changes with LPS-induced inflammation and apolipoprotein A-I and A-II transgenes

We present reference maps of the mouse serum proteome (run under reducing and non-reducing conditions), from control animals, from mice injected with lipopolysaccharide (LPS) to induce systemic inflammation, and from mice transgenic for human apolipoproteins A-I and A-II. Seventy-seven spots/spot chains from the reducing gels were identified by HPLC MS/MS, representing 28 distinct proteins, including a species-specific protease inhibitor, contrapsin, and high levels of carboxylesterase. The concentrations of acute-phase reactants were monitored for 96 h after LPS challenge. The greatest changes (four-fold 48 h after LPS administration) were observed for haptoglobin and hemopexin. Orosomucoid/alpha(1)-acid glycoprotein and apolipoprotein A-I increased steadily, to 50-60% above baseline at 96 h from stimulation. In mice transgenic for human apolipoprotein A-I the levels of expression of orosomucoid/alpha(1)-acid glycoprotein, alpha(1)-macroglobulin, esterase, kininogen and contrapsin were altered compared to knockout mice lacking apolipoprotein A-I. In contrast, except for the presence of apolipoprotein A-II, no statistically significant difference was observed in mice transgenic for human apolipoprotein A-II.     

Characterization of the human salivary proteome by capillary isoelectric focusing/nanoreversed-phase liquid chromatography coupled with ESI-tandem MS

Saliva is a readily available body fluid with great diagnostic potential. The foundation for saliva-based diagnostics, however, is the development of a complete catalog of secreted and "leaked" proteins detectable in saliva. By employing a capillary isoelectric focusing-based multidimensional separation platform coupled with electrospray ionization tandem mass spectrometry (MS), a total of 5338 distinct peptides were sequenced, leading to the identification of 1381 distinct proteins. A search of bacterial protein sequences also identified many peptides unique to several organisms and unique to the NCBI nonredundant database. To the best of our knowledge, this proteome study represents the largest catalog of proteins measured from a single saliva sample to date. Data analysis was performed on individual MS/MS spectra using the highly specific peptide identification algorithm, OMSSA. Searches were conducted against a decoyed SwissProt human database to control the false-positive rate at 1%. Furthermore, the well-curated SwissProt sequences represent perhaps the least redundant human protein sequence database (12,484 records versus the 50,009 records found in the International Protein Index human database), therefore minimizing multiple protein inferences from single peptides. This combined bioanalytical and bioinformatic approach has established a solid foundation for building up the human salivary proteome for the realization of the diagnostic potential of saliva.     

Interaction between calcofluor white and carbohydrates of alpha 1-acid glycoprotein.

Interactions between the fluorescent probe, calcofluor white, and human serum albumin (HSA) and alpha 1-acid glycoprotein (orosomucoid) are compared. The two proteins have comparable isoelectric points, but alpha 1-acid glycoprotein is highly glycosylated (40% of glycans by weight), while the serum albumin is not. Binding of calcofluor to the proteins induces an increase in both the fluorescence anisotropy and the fluorescence intensity of the fluorophore. Also, we found that the calcofluor exhibits a fluorescence emission with a maximum located at 432, 415 or 445 nm, respectively, in the absence of proteins, in the presence of HSA, and in the presence of alpha 1-acid glycoprotein. The stoichiometries of the calcofluor-serum albumin and calcofluor-alpha 1-acid glycoprotein complexes are 2:1 and 1:1, respectively. The association constants are 0.04 and 0.15 microM-1, respectively. The calcofluor does not interact with Lens culinaris agglutinin (LCA), although the protein has a hydrophobic site. Nevertheless, one cannot exclude that the binding of the fluorophore to the HSA is nonspecific. Our results, when compared with those obtained with calcofluor dissolved in the hydrophobic solvent isobutanol, and with the fluorescent probe, potassium 6-(p-toluidino)-2-naphthalenesulfonate (TNS), bound to alpha 1-acid glycoprotein, indicate that the emission of calcofluor bound to HSA occurs from a hydrophobic state, while that of calcofluor bound to alpha 1-acid glycoprotein occurs from a hydrophilic state. The fluorescence intensity of calcofluor decreases in the presence of carbohydrates isolated from alpha 1-acid glycoprotein, while it increases in the presence of alpha 1-cellulose. Thus, calcofluor interacts mainly with the glycan moiety of alpha 1-acid glycoprotein, and its fluorescence is sensitive to the secondary structure of the glycans.     

Interaction between calcofluor white and carbohydrates of alpha 1-acid glycoprotein.

Interactions between the fluorescent probe, calcofluor white, and human serum albumin (HSA) and alpha 1-acid glycoprotein (orosomucoid) are compared. The two proteins have comparable isoelectric points, but alpha 1-acid glycoprotein is highly glycosylated (40% of glycans by weight), while the serum albumin is not. Binding of calcofluor to the proteins induces an increase in both the fluorescence anisotropy and the fluorescence intensity of the fluorophore. Also, we found that the calcofluor exhibits a fluorescence emission with a maximum located at 432, 415 or 445 nm, respectively, in the absence of proteins, in the presence of HSA, and in the presence of alpha 1-acid glycoprotein. The stoichiometries of the calcofluor-serum albumin and calcofluor-alpha 1-acid glycoprotein complexes are 2:1 and 1:1, respectively. The association constants are 0.04 and 0.15 microM-1, respectively. The calcofluor does not interact with Lens culinaris agglutinin (LCA), although the protein has a hydrophobic site. Nevertheless, one cannot exclude that the binding of the fluorophore to the HSA is nonspecific. Our results, when compared with those obtained with calcofluor dissolved in the hydrophobic solvent isobutanol, and with the fluorescent probe, potassium 6-(p-toluidino)-2-naphthalenesulfonate (TNS), bound to alpha 1-acid glycoprotein, indicate that the emission of calcofluor bound to HSA occurs from a hydrophobic state, while that of calcofluor bound to alpha 1-acid glycoprotein occurs from a hydrophilic state. The fluorescence intensity of calcofluor decreases in the presence of carbohydrates isolated from alpha 1-acid glycoprotein, while it increases in the presence of alpha 1-cellulose. Thus, calcofluor interacts mainly with the glycan moiety of alpha 1-acid glycoprotein, and its fluorescence is sensitive to the secondary structure of the glycans.     

Protein glycosylation regulates the externalization of two distinct classes of glucocorticoid-induced glycoproteins in rat hepatoma cells

The relationship of protein glycosylation to the externalization of glucocorticoid inducible alpha1-acid glycoprotein and mouse mammary tumor virus glycoproteins was examined in M1.54, a clonal population of mouse mammary tumor virus-infected rat hepatoma cells. Multiple freeze-thaw of isolated microsomes revealed that while alpha 1-acid glycoprotein is carried through the cell as a soluble component of vesicles, extracellular viral glycoproteins are initially membrane-associated. At concentrations of tunicamycin that specifically inhibited N-linked protein glycosylation, alpha 1-acid glycoprotein fractionated between the cellular and extracellular compartments. Thus, approximately one half of the newly synthesized, nonglycosylated (22,000 Mr) alpha 1-acid glycoprotein was rapidly secreted with kinetics similar to its glycosylated counterpart (release half-time of 60 min), while the remaining species first localized in an undefined intracellular compartment prior to its slow secretion (release half-time of 24 h). The same distribution of nonglycosylated alpha 1-acid glycoprotein was observed at various absolute levels of polypeptide, suggesting that this was not due simply to the saturation of an efficient secretory pathway at high polypeptide levels. In contrast to alpha 1-acid glycoprotein, no labeled viral antigens were released by tunicamycin-treated M1.54, while a nonglycosylated viral precursor glycopolyprotein was expressed intracellularly. Taken together, these results suggest that carbohydrate attachment strongly regulates the externalization of both alpha 1-acid glycoprotein and mouse mammary tumor virus species, which represent two distinct classes of extracellular glycoproteins.     

Acute phase protein alpha 1-acid glycoprotein interacts with plasminogen activator inhibitor type 1 and stabilizes its inhibitory activity.

alpha(1)-Acid glycoprotein, one of the major acute phase proteins, was found to interact with plasminogen activator inhibitor type 1 (PAI-1) and to stabilize its inhibitory activity toward plasminogen activators. This conclusion is based on the following observations: (a) alpha(1)-acid glycoprotein was identified to bind PAI-1 by a yeast two-hybrid system. Three of 10 positive clones identified by this method to interact with PAI-1 contained almost the entire sequence of alpha(1)-acid glycoprotein; (b) this protein formed complexes with PAI-1 that could be immunoprecipitated from both the incubation mixtures and blood plasma by specific antibodies to either PAI-1 or alpha(1)-acid glycoprotein. Such complexes could be also detected by a solid phase binding assay; and (c) the real-time bimolecular interactions monitored by surface plasmon resonance indicated that the complex of alpha(1)-acid glycoprotein with PAI-1 is less stable than that formed by vitronectin with PAI-1, but in both cases, the apparent K(D) values were in the range of strong interactions (4.51 + 1.33 and 0.58 + 0.07 nm, respectively). The on rate for binding of PAI-1 to alpha(1)-glycoprotein or vitronectin differed by 2-fold, indicating much faster complex formation by vitronectin than by alpha(1)-acid glycoprotein. On the other hand, dissociation of PAI-1 bound to vitronectin was much slower than that from the alpha(1)-acid glycoprotein, as indicated by 4-fold lower k(off) values. Furthermore, the PAI-1 activity toward urokinase-type plasminogen activator and tissue-type plasminogen activator was significantly prolonged in the presence of alpha(1)-acid glycoprotein. These observations suggest that the complex of PAI-1 with alpha(1)-acid glycoprotein can play a role as an alternative reservoir of the physiologically active form of the inhibitor, particularly during inflammation or other acute phase reactions.     

Involvement of various organs in the initial plasma clearance of differently glycosylated rat liver secretory proteins

The initial plasma clearance and organ distribution of alpha 1-acid glycoprotein and alpha 2-macroglobulin carrying different types of oligosaccharide, side chains was studied in rats. The differently glycosylated proteins were synthesized by rat hepatocytes in culture in the presence of tunicamycin (unglycosylated form), swainsonine (hybrid type), or 1-deoxymannojirimycin (high-mannose type). Deglycosylated glycoproteins (Asn-GlcNAc) were obtained by endoglucosaminidase H treatment of high-mannose-type glycoproteins. Ten minutes after intravenous injection 3% of complex type, 26% of hybrid type, 84% of high-mannose type. 64% of unglycosylated and 80% of deglycosylated alpha 1-acid glycoprotein disappeared from the plasma. The respective values for alpha 2-macroglobulin were 26%, 42%, 59% and 67%. When the clearance of total hepatic secretory proteins was examined, major differences between glycosylated and unglycosylated (glyco)proteins were found, particularly in the case of low-molecular-mass polypeptides. Whereas complex-type alpha 1-acid glycoprotein and alpha 2-macroglobulin showed no accumulation in various organs, hybrid-type alpha 1-acid glycoprotein and alpha 2-macroglobulin were present in spleen and liver. High-mannose-type alpha 1-acid glycoprotein and alpha 2-macroglobulin also accumulated mainly in spleen and liver. Spleen had the highest specific activity; liver, due to its larger organ mass, represented the major organ for the uptake of high-mannose-type glycoproteins. Competition experiments with mannan and GlcNAc-bovine-serum-albumin showed a mannose/GlcNAc receptor-mediated removal. Whereas unglycosylated alpha 1-acid glycoprotein was taken up by the kidney, unglycosylated alpha 2-macroglobulin was found in the spleen. Deglycosylated glycoproteins (Asn-GlcNAc) were removed from the plasma via two different mechanisms: firstly, clearance by the kidney similar to the unglycosylated glycoproteins; secondly, clearance by a mannose/GlcNAc receptor-mediated uptake mainly into the spleen. We conclude that N-linked oligosaccharide side chains are important for the plasma survival of hepatic secretory glycoproteins and that unphysiologically glycosylated forms are cleared by different mechanisms.     

Toward a standardized saliva proteome analysis methodology

The present study aimed the evaluation of saliva sample pre-treatment, in particular the sample clearance usually performed by centrifugation, to the contribution of salivary proteome and peptidome. Using in-gel and off-gel approaches, a large content of salivary proteins was detected in the pellet fraction that is usually discarded. In addition, chaotropic/detergent treatment in combination with sonication, before the centrifugation step, resulted in salivary complex disruption and consequently in the extraction of high amounts of proteins. Based on this data, we suggest the use of urea/detergent with sonication as a standard saliva sample pre-treatment procedure. We also described a procedure to extract salivary peptides which can be performed even after saliva sample treatment with chaotropic/detergents. In overall, we reported for the first time the contribution of the pellet fraction to the whole saliva proteome. iTRAQ analysis highlighted a higher number of different peptides as well as distinct quantities of each protein class when after sample treatment with urea and sonication, acetone precipitation followed by solubilization with acetonitrile/HCl was performed.     

Saliva proteome research: current status and future outlook

Human saliva harbours proteins of clinical relevance and about 30% of blood proteins are also present in saliva. This highlights that saliva can be used for clinical applications just as urine or blood. However, the translation of salivary biomarker discoveries into clinical settings is hampered by the dynamics and complexity of the salivary proteome. This review focuses on the current status of technological developments and achievements relating to approaches for unravelling the human salivary proteome. We discuss the dynamics of the salivary proteome, as well as the importance of sample preparation and processing techniques and their influence on downstream protein applications; post-translational modifications of salivary proteome and protein: protein interactions. In addition, we describe possible enrichment strategies for discerning post-translational modifications of salivary proteins, the potential utility of selected-reaction-monitoring techniques for biomarker discovery and validation, limitations to proteomics and the biomarker challenge and future perspectives. In summary, we provide recommendations for practical saliva sampling, processing and storage conditions to increase the quality of future studies in an emerging field of saliva clinical proteomics. We propose that the advent of technologies allowing sensitive and high throughput proteome-wide analyses, coupled to well-controlled study design, will allow saliva to enter clinical practice as an alternative to blood-based methods due to its simplistic nature of sampling, non-invasiveness, easy of collection and multiple collections by untrained professionals and cost-effective advantages.     

Saliva proteome profiling reveals potential salivary biomarkers for detection of oral cavity squamous cell carcinoma

Oral cavity squamous cell carcinoma (OSCC), which is frequently associated with poor prognosis and mortality, is a leading cause of cancer‐related death worldwide. Discovery of body fluid accessible biomarkers is needed to improve OSCC screening. To this end, we profiled proteomes of saliva from the healthy volunteers, the individuals with oral potentially malignant disorders (OPMD), and the OSCC patients by means of SDS‐PAGE coupled with LC‐MS/MS. In the control, the OPMD, and the OSCC groups, 958, 845, and 1030 salivary proteins were detected, respectively. With spectral counting‐based label‐free quantification, 22 overexpressed salivary proteins were identified in the OSCC group compared with the healthy controls and the OPMD individuals. Among them, resistin (RETN) was subjected to further validation with an independent cohort using ELISA. The data confirmed that the salivary RETN levels in the OSCC patients were significantly higher than that in the healthy or in the OPMD group. Moreover, the elevated levels of salivary RETN were highly correlated with late‐stage primary tumors, advanced overall stage, and lymph‐node metastasis. Our results not only reveal that profiling of saliva proteome is feasible for discovery of OSCC biomarkers, but also identify RETN as a potential salivary biomarker for OSCC detection.     

Studies on incorporation of mannose into rat alpha 1-acid glycoprotein: evidence for precursor forms in rough membranes

Rats were given pulse injections of D-[14C]mannose and were killed at various times up to 60 min after injection. Rough, smooth, and Golgi fractions were prepared from liver, and alpha 1-acid glycoprotein was isolated from Lubrol extracts of the fractions. The kinetics of incorporation of D-[14C]mannose into total protein, Lubrol protein, and alpha 1-acid glycoprotein showed that proteins associated with rough fractions had particularly high specific radioactivities at early times of incorporation. One explanation for the kinetic data is that glycoproteins contain a high mannose content at early times of assembly of oligosaccharide chains. This idea was confirmed in the case of alpha 1-acid glycoprotein by isolation of a high mannose containing precursor species of alpha 1-acid glycoprotein from rough fractions of liver. This species contained 56 residues of hexose (mainly mannose) compared with 35 residues of hexose (roughly equal amounts of mannose and galactose) which are found in the native protein. It is proposed that the high mannose precursor is a form of alpha 1-acid glycoprotein that exists at an early stage in assembly of the glycoprotein and which contains largely unprocessed carbohydrate chains. In addition, evidence is presented from amino acid analyses and gel electrophoresis of the high mannose precursor and another fraction from which it is formed by limited tryptic treatment, that pro-forms of alpha 1-acid glycoprotein with extensions of the polypeptide chain may also exist.     

The scientific exploration of saliva in the post-proteomic era: from database back to basic function

The proteome of human saliva can be considered as being essentially completed. Diagnostic markers for a number of diseases have been identified among salivary proteins and peptides, taking advantage of saliva as an easy-to-obtain biological fluid. Yet, the majority of disease markers identified so far are serum components and not intrinsic proteins produced by the salivary glands. Furthermore, despite the fact that saliva is essential for protecting the oral integuments and dentition, little progress has been made in finding risk predictors in the salivary proteome for dental caries or periodontal disease. Since salivary proteins, and in particular the attached glycans, play an important role in interactions with the microbial world, the salivary glycoproteome and other post-translational modifications of salivary proteins need to be studied. Risk markers for microbial diseases, including dental caries, are likely to be discovered among the highly glycosylated major protein species in saliva. This review will attempt to raise new ideas and also point to under-researched areas that may hold promise for future applicability in oral diagnostics and prediction of oral disease.     

Large-scale identification of proteins in human salivary proteome by liquid chromatography/mass spectrometry and two-dimensional gel electrophoresis-mass spectrometry

Human saliva contains a large number of proteins and peptides (salivary proteome) that help maintain homeostasis in the oral cavity. Global analysis of human salivary proteome is important for understanding oral health and disease pathogenesis. In this study, large-scale identification of salivary proteins was demonstrated by using shotgun proteomics and two-dimensinal gel electrophoresis-mass spectrometry (2-DE-MS). For the shotgun approach, whole saliva proteins were prefractionated according to molecular weight. The smallest fraction, presumably containing salivary peptides, was directly separated by capillary liquid chromatography (LC). However, the large protein fractions were digested into peptides for subsequent LC separation. Separated peptides were analyzed by on-line electrospray tandem mass spectrometry (MS/MS) using a quadrupole-time of flight mass spectrometer, and the obtained spectra were automatically processed to search human protein sequence database for protein identification. Additionally, 2-DE was used to map out the proteins in whole saliva. Protein spots 105 in number were excised and in-gel digested; and the resulting peptide fragments were measured by matrix-assisted laser desorption/ionization-mass spectrometry and sequenced by LC-MS/MS for protein identification. In total, we cataloged 309 proteins from human whole saliva by using these two proteomic approaches.     

Cross‐species comparison of mammalian saliva using an LC–MALDI based proteomic approach

Despite the importance of saliva in the regulation of oral cavity homeostasis, few studies have been conducted to quantitatively compare the saliva of different mammal species. Aiming to define a proteome signature of mammals’ saliva, an in‐depth SDS‐PAGE–LC coupled to MS/MS (GeLC–MS/MS) approach was used to characterize the saliva from primates (human), carnivores (dog), glires (rat and rabbit), and ungulates (sheep, cattle, horse). Despite the high variability in the number of distinct proteins identified per species, most protein families were shared by the mammals studied with the exception of cattle and horse. Alpha‐amylase is an example that seems to reflect the natural selection related to digestion efficacy and food recognition. Casein protein family was identified in all species but human, suggesting an alternative to statherin in the protection of hard tissues. Overall, data suggest that different proteins might assure a similar role in the regulation of oral cavity homeostasis, potentially explaining the specific mammals’ salivary proteome signature. Moreover, some protein families were identified for the first time in the saliva of some species, the presence of proline‐rich proteins in rabbit's saliva being a good example.     

A distinct repertoire of autoantibodies in hepatocellular carcinoma identified by proteomic analysis

Chronic infections with hepatitis B (HBV) and hepatitis C (HCV) viruses are major risk factors for hepatocellular carcinoma (HCC). We have utilized a proteomic approach to determine whether a distinct repertoire of autoantibodies can be identified in HCC. Sera from 37 patients with HCC and 31 subjects chronically infected with HBV or HCV without HCC were investigated. Sera from 116 patients with other cancers, three patients with systemic lupus erythematosus, and 24 healthy subjects were utilized as controls. We report the identification of eight proteins, for each of which autoantibodies were detected in sera from more than 10% of patients with HCC but not in sera from healthy individuals (p < 0.05). Autoantibodies to four of these proteins were detected at a comparable frequency in sera from patients with chronic hepatitis. The other four proteins, which consisted of calreticulin isoforms, cytokeratin 8, nucleoside diphosphate kinase A, and F(1)-ATP synthase beta-subunit, induced autoantibodies among patients with HCC, independently of their HBV/HCV status. Calreticulin, and a novel truncated form of calreticulin (Crt32) we have identified, most commonly elicited autoantibodies among patients with HCC (27%). We conclude that a distinct repertoire of autoantibodies is associated with HCC that may have utility in early diagnosis of HCC among high risk subjects with chronic hepatitis.     

Quantitative proteomic analysis of the fall armyworm saliva

Lepidopteran larvae secrete saliva on plant tissues during feeding. Components in the saliva may aid in food digestion, whereas other components are recognized by plants as cues to elicit defense responses. Despite the ecological and economical importance of these plant-feeding insects, knowledge of their saliva composition is limited to a few species. In this study, we identified the salivary proteins of larvae of the fall armyworm (FAW), Spodoptera frugiperda; determined qualitative and quantitative differences in the salivary proteome of the two host races—corn and rice strains—of this insect; and identified changes in total protein concentration and relative protein abundance in the saliva of FAW larvae associated with different host plants. Quantitative proteomic analyses were performed using labeling with isobaric tags for relative and absolute quantification followed by liquid chromatography-tandem mass spectrometry. In total, 98 proteins were identified (>99% confidence) in the FAW saliva. These proteins were further categorized into five functional groups: proteins potentially involved in (1) plant defense regulation, (2) herbivore offense, (3) insect immunity, (4) detoxification, (5) digestion, and (6) other functions. Moreover, there were differences in the salivary proteome between the FAW strains that were identified by label-free proteomic analyses. Thirteen differentially identified proteins were present in each strain. There were also differences in the relative abundance of eleven salivary proteins between the two FAW host strains as well as differences within each strain associated with different diets. The total salivary protein concentration was also different for the two strains reared on different host plants. Based on these results, we conclude that the FAW saliva contains a complex mixture of proteins involved in different functions that are specific for each strain and its composition can change plastically in response to diet type.     

Salivary protein profiling in type I diabetes using two-dimensional electrophoresis and mass spectrometry

Owing to its noninvasive collection, saliva is considered as a potent diagnostic fluid. The goal of this study was to investigate the modification of the salivary proteome occurring in type 1 diabetes to highlight potential biomarkers of the pathology. High-resolution two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were combined to perform a largescale analysis. The proteomic comparison of saliva samples from healthy subjects and poorly controlled type 1 diabetes patients revealed a modulation of 23 proteins. Fourteen isoforms of α-amylase, one prolactin inducible protein, three isoforms of salivary acidic protein-1, and three isoforms of salivary cystatins SA-1 were detected as under expressed, whereas two isoforms of serotransferrin were over expressed in the pathological condition. The proteins under expressed were all known to be implicated in the oral anti-inflammatory process, suggesting that the pathology induced a decrease of non-immunological defense of oral cavity. As only particular isoforms of proteins were modulated, type 1 diabetes seemed to differentially affect posttranslational modification. Authors have contributed equally to this work.