Abnormal Galactosylated–Glycans recognized by Bandeiraea Simplicifolia Lectin I in saliva of patients with breast Cancer

Currently, the definitive diagnosis in breast cancer requires biopsy and histopathology, such the most effective markers are tissue-based. However, the advantages of saliva in collection and storage make it possible for assessing human pathology and contributing to the development of cancer-related biomarkers for clinical application. The present study validated alteration of salivary protein glycopatterns recognized by Bandeiraea simplicifolia lectin I (BS-I) in the saliva of patients with breast diseases using saliva microarrays, and the N/O-glycan profiles of their salivary glycoproteins isolated by the BS-I-magnetic particle conjugates from 259 female subjects (66 healthy volunteers (HV), 65 benign breast cyst or tumor patients (BB), 66 patients with breast cancer in stage I (BC-I) and 62 patients with breast cancer in stage II (BC-II)) were analyzed by MALDI-TOF/TOF-MS. The results showed that the expression level of galactosylated glycans recognized by BS-I was significantly increased in patients with breast cancer compared with HV (p < 0.05). Totally, there were 11/10, 10/19, 7/24 and 7/9 galactosylated N-/O-linked glycans were identified and annotated from the pooled salivary samples of HV, BB, BC-I and BC-II, respectively. One galactosylated N-glycan peak (m/z 2773.977), and 4 galactosylated O-glycan peaks (m/z 868.295, 882.243, 884.270 and 1030.348) were found only in BC-I. These findings could provide pivotal information on galactosylated N/O-linked glycans related to breast cancer, and promote the study of biomarkers for early-stage breast cancer based on precise alterations of galactosylated N/O-glycans in saliva.     

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.     

Alteration of liver glycopatterns during cirrhosis and tumor progression induced by HBV

The incidence of hepatocellular carcinoma (HCC) is closely correlated with hepatitis B virus (HBV)-induced liver cirrhosis. Structural changes in the glycans of serum and tissue proteins are reliable indicators of liver damage. However, little is known about the alteration of liver glycopatterns during cirrhosis and tumor progression induced by HBV infection. This study compared the differential expression of liver glycopatterns in 7 sets of normal pericarcinomatous tissues (PCTs), cirrhotic, and tumor tissues from patients with liver cirrhosis and HCC induced by HBV using lectin microarrays. Fluorescence-based lectin histochemistry and lectin blotting were further utilized to validate and assess the expression and distribution of certain glycans in 9 sets of corresponding liver tissue sections. Eight lectins (e.g., Jacalin and AAL) revealed significant difference in cirrhotic tissues versus PCTs. Eleven lectins (e.g., EEL and SJA) showed significant alteration during cirrhotic and tumor progression. The expression of Galα1-3(Fucα1-2)Gal (EEL) and fucosyltransferase 1 was mainly increasing in the cytoplasm of hepatocytes during PCTs-cirrhotic-tumor tissues progression, while the expression of T antigen (ACA and PNA) was decreased sharply in cytoplasm of tumor hepatocytes. Understanding the precision alteration of liver glycopatterns related to the development of hepatitis, cirrhosis, and tumor induced by HBV infection may help elucidate the molecular mechanisms underlying the progression of chronic liver diseases and develop new antineoplastic therapeutic strategies.     

Analysis of oral microbial community and Th17‐associated cytokines in saliva of patients with oral lichen planus

Oral lichen planus (OLP) is a chronic inflammatory disorder of oral mucosa of unknown cause. Microbial infection and dysimmunity appear to play important roles in its pathogenesis. In this study, differences in genetic profiling of salivary microbial communities in two subtypes of OLP and healthy controls were evaluated by means of PCR‐denaturing gradient gel electrophoresis (DGGE). Additionally, ELISA was used to investigate the possible role of Th17 in lesion formation by detecting two related cytokines IL‐17 and IL‐23 in the saliva of OLP patients. When the DGGE profiles were analyzed, the bacterial populations were found to be significantly less rich in subjects with reticular and erosive OLP than in healthy controls. There was significantly less microbial diversity, as denoted by the Shannon index, in saliva samples from subjects with erosive OLP than in those from healthy controls. Cluster analysis and principal component analysis showed that the DGGE profiles formed distinctly group‐specific clusters. Salivary concentrations of IL‐17 in subjects with erosive OLP group were significantly higher than in those with reticular OLP and healthy controls. What's more, significantly positive correlations were observed between salivary IL‐17 concentrations and disease clinical scores. Microbial richness and diversity was negatively correlated with salivary IL‐17 concentrations. These results suggest there is significantly less salivary bacterial diversity and complexity in subjects with OLP han in healthy controls and that the shifted community composition is closely related to an immune cytokine, IL‐17.     

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.     

Possible salivary and serum biomarkers for oral lichen planus

There are few reports concerning the potential for clinical application of oxidative stress (OS) and collagen degradation markers in oral lichen planus (OLP) patients. We investigated the possibility of using some disease-related biomarkers in saliva and serum of OLP patients. Our study included 30 patients with OLP and 30 controls. We evaluated serum and salivary OS biomarkers including 8-OHdG, MDA, uric acid, TAC and GPx. We also investigated collagen degradation markers such as CTX I and MMP-8. We found significantly increased salivary levels of MMP-8 and CTX I in the OLP group compared to controls and significant differences between the OLP and control groups in serum and saliva for 8-OHdG, MDA (significantly increased), uric acid, TAC and GPx (significantly reduced). Currently there are no criteria for evaluating which OLP patients have a greater risk of malignant transformation. In addition to clinical surveillance, the serum and salivary biomarkers that we evaluated may be useful biomarkers for monitoring OLP patients in the future.     

Comparison of the Antimicrobial Adhesion Potential of Human Body Fluid Glycoconjugates Using Fucose-Binding Lectin (PA-IIL) of Pseudomonas aeruginosa and Ulex europaeus Lectin (UEA-I)

Pseudomonas aeruginosa produces a fucose-binding lectin (PA-IIL) which strongly binds to human cells. This lectin was shown to be highly sensitive to inhibition by fucose-bearing human milk glycoproteins. Since the glycans of these glycoproteins mimic human cell receptors, they may function as decoys in blocking lectin-dependent pathogen adhesion to the host cells. Human saliva and seminal fluid also contain such compounds, and body fluids of individuals who are “secretors” express additional fucosylated (alpha 1,2) residues. The latter are selectively detected by Ulex europaeus lectin UEA-I. The aim of the present research was to compare the PA-IIL and UEA-I interactions with human salivas and seminal fluids of “secretors” and “nonsecretors” with those obtained with the respective milks. Using hemagglutination inhibition and Western blot analyses, we showed that PA-IIL interactions with the saliva and seminal fluid glycoproteins were somewhat weaker than those obtained with the milk and that “nonsecretor” body fluids were not less efficient than those of “secretors” in PA-IIL blocking. UEA-I, which interacted only with the “secretors” glycoproteins, was most sensitive to those of the seminal fluids.     

A comparison of native and non-urate Total Antioxidant Capacity of fasting plasma and saliva among middle-aged and older subjects

Objectives: As plasma and salivary total antioxidant capacity (TAC) is mainly contributed by uric acid (UA), the present study measures non-urate TAC (Nu-TAC). The aim of the study was to correlate plasma native TAC, Nu-TAC and UA with their salivary analogues, and compare the UA contribution in both body fluids using two different methods.

Methods: The study involved 55 middle-aged and older subjects (66.7?±?4.5 years). TAC was determined simultaneously with two methods (ferric reducing ability of plasma – FRAP, 2.2-diphenyl-1-picryl-hydrazyl – DPPH and countertypes for saliva – FRAS and DPPHS test), with and without UA (native TAC and Nu-TAC, respectively). Plasma UA and salivary UA (SUA) were assessed.

Results: Subjects with increased FRAP, DPPH and UA had higher FRAS, DPPHS and SUA, respectively (P?P?Discussion: Our findings suggest that saliva is a good predictor for native plasma TAC but not for Nu-TAC. UA level is comparably dominant in saliva and in plasma according to DPPH, but lower in plasma according to FRAP.     

Oxidative Modulation of the Glutathione-redox Couple Enhances Lipopolysaccharide-induced Interleukin 12 P40 Production by a Mouse Macrophage Cell Line,J774A.1

Abstract

Reactions of salivary nitrite with components of wine were studied using an acidic mixture of saliva and wine. The formation of nitric oxide (NO) in the stomach after drinking wine was observed. The formation of NO was also observed in the mixture (pH 3.6) of saliva and wine, which was prepared by washing the oral cavity with wine. A part of the NO formation in the stomach and the oral cavity was due to the reduction of salivary nitrite by caffeic and ferulic acids present in wine. Ethyl nitrite produced by the reaction of salivary nitrite and ethyl alcohol in wine also contributed to the formation of NO. In addition to the above reactions, caffeic acid in wine could be transformed to the oxathiolone derivative, which might have pharmacological functions. The results obtained in this study may help in understanding the effects of drinking wine on human health.     

Salivary levels of tumor necrosis factor-alpha in oral lichen planus

OBJECTIVE: Oral lichen planus (OLP) is chronic inflammatory disease of the oral mucosa, presenting in various clinical forms. The etiology of OLP is still unknown but mounting evidence points to the immunologic basis of this disorder. AIM: Our study was undertaken to quantify the salivary levels of pro-inflammatory tumor necrosis factor-alpha (TNF-alpha) in the reticular and the erosive/atrophic forms of OLP, compared with age-matched healthy control volunteers. SUBJECTS AND METHODS: Whole saliva from 40 patients with active lesions of OLP, as well as from 20 healthy persons, was investigated for the presence of TNF-alpha by enzyme immunoassay. RESULTS: Salivary TNF-alpha levels were significantly increased in patients with OLP in comparison with healthy subjects. The presence of TNF-alpha showed positive correlation to clinical forms of OLP, being significantly higher in the erosive/atrophic type than in the reticular type of disease. CONCLUSION: Saliva provides an ideal medium for the detection of pro-inflammatory markers of the oral cavity. In patients with OLP, TNF-alpha levels in saliva are elevated, correlating with the severity of illness. Salivary TNF-alpha analysis may be a useful diagnostic tool and a potential prognostic marker in OLP.     

The role of the saliva antioxidant barrier to reactive oxygen species with regard to caries development

Objectives: The aim of this study was to evaluate the role of the antioxidant barrier in the saliva of children with caries, and its impact on the colonization of cariogenic bacteria.

Methods: This is a cross-sectional study of 81 children aged 1–5 years. Antioxidant levels and salivary bacterial profiles were measured. Patients were divided into two groups as follows: initial stage decay, termed non-cavitated (1–2 in International Caries Detection and Assessment System (ICDAS)), and extensive decay, termed cavitated lesions (5–6 in ICDAS). The control group includes children without caries.

Results: The linear regression model demonstrated that the GSH, GSSG, GSH/GSSG, and total antioxidant capacity levels are influenced (P?P?Discussion: Our results indicate that the high levels of antioxidants in saliva increase significantly in children in line with the salivary cariogenic bacterial profiles and caries progression.     

Glycosylation in viral hepatitis

BackgroundThe interaction between hepatitis viruses and host cells is regulated by glycans exposed on the surfaces of human and viruses cells. As the biosynthesis and degradation of human glycoproteins take place at the highest level in the liver, the changes in glycosylation of serum proteins may potentially be useful in the diagnosis of liver pathology. On the other hand, specific alterations in viruses envelope glycans could cause large changes in the entry process of hepatitis viruses into a host cells.Scope of reviewUnique alterations in glycosylation of specific proteins can be detected in HBV and HCV infected patients especially with confirmed fibrosis/cirrhosis. On the other hand, viral envelope proteins that bind to host cells are glycosylated. These glycosylated proteins play a key role in recognition, binding and penetration of the host cells. In this review we summarized the knowledge about significance of glycosylation for viral and host factors.Major conclusionsGlycosylation changes in single serum glycoproteins are noticed in the sera of patients with viral hepatitis. However, a more specific biomarker for the diagnosis of chronic hepatitis than that of a single glycosylated molecule is systemic investigation of complete set of glycan structures (N-glycome). Glycans play important roles in the viral biology cycle especially as a connecting element with host receptors.General significanceThe interaction between virus glycoproteins and cellular receptors, which are also glycoproteins, determines the possibility of virus penetration into host cells. Therefore these glycans can be the targets for the developing of novel treatment strategies of viral hepatitis.     

NF-kappaB-dependent cytokines in saliva and serum from patients with oral lichen planus: a study in an ethnic Chinese population

To access the potential roles of NF-kappaB-dependent pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha), interleukin-6(IL-6), and interleukin-8(IL-8), in oral lichen planus (OLP), and to explore whether salivary detection of these cytokines is a better monitoring tool than serum for OLP, 30 Chinese patients with OLP and 30 age-sex-matched controls were enrolled in this study. Salivary and serum levels of cytokines were screened by enzyme immunoassay, and both salivary and serum levels of these cytokines were significantly increased in OLP patients in comparison with healthy subjects. Positive statistical correlations existed between these cytokines in serum and saliva. With regards to the subtypes of this illness, only salivary IL-8 levels in erosive form were remarkably inclined than that in reticular form. The results suggest that saliva-based test may provide a more cost-effective adjunctive tool for the detection of pro-inflammatory cytokines in OLP, and salivary IL-8 analysis may be a promising biomarker for OLP severity.     

The antioxidant effects of pomegranate extract on local and remote organs in a mesenteric ischemia and reperfusion model

Objectives: We investigated whether pomegranate extract plays a protective antioxidant role against mesenteric ischemia–reperfusion injury (IR), which can lead to a systemic response and damage distant organs, such as the lung, liver, and kidney.

Methods: Forty female Wistar–Albino rats were separated into four groups: laparotomy, laparotomy?+?PG, mesenteric IR, and mesenteric IR and pomegranate (IR?+?PG). In the laparotomy?+?PG and IR?+?PG groups, pomegranate (225?mg/kg) was given by oral gavage at the beginning of the study. Ischemia was induced for 30 minutes, and reperfusion was subsequently allowed for 60 minutes in the IR and IR?+?PG groups. The malondialdehyde (MDA) and total antioxidant activity (AOA) levels were evaluated in blood samples. Additionally, all tissues were removed for the measurement of AOA and total oxidant status as well as for subsequent histopathological evaluation. The oxidative stress index was calculated.

Results: Histopathological changes in all organs were significantly higher in the IR group and significantly lower in the IR?+?PG group vs. the other groups. Serum MDA levels were significantly lower in the IR?+?PG group than in the IR group. No significant difference was found in AOA levels of the groups.

Discussion: These data may explain the positive protective effects of pomegranate based on the histopathologic findings in ischemic conditions in an intestinal IR injury model.     

Variability of salivary markers of oxidative stress and antioxidant status in young healthy individuals

Objectives: Salivary advanced glycation end-products (AGEs), advanced oxidation protein products (AOPP), total antioxidant capacity (TAC), and ferric reducing ability of saliva (FRAS) are increased in various diseases. Little data exist for these markers in the healthy population. The aim of this study was to assess the inter-individual and intra-individual variability of AGEs, AOPP, TAC, and FRAS in the saliva of young healthy individuals.

Methods: Unstimulated saliva samples were collected from 16 females and 18 males daily over a period of 30 days. Markers were measured using spectrophotometric and spectrofluorometric microplate-based methods.

Results: All salivary markers measured were significantly higher in men than in women (P?<?0.05 for AGEs; P?<?0.001 for AOPP, TAC, and FRAS). The inter-individual variability was approximately 60% for AGEs and AOPP and 30–40% for TAC and FRAS in both genders. The inter-individual variability of FRAS was higher in men vs. women (P?<?0.01). Intra-individual variability ranged from 20% for TAC, to 30% for AGES and FRAS and 45% for AOPP.

Discussion: Intra-individual variability of salivary AGEs, AOPP, TAC, and FRAS indicates that their use is currently limited to large cohort studies. Identifying the underlying factors related to the high inter-individual and intra-individual variability is needed. Sex differences should be considered in future studies.     

Selective isolation and analysis of glycoprotein fractions and their glycomes from hepatocellular carcinoma sera

As one of the most important post‐translational modifications, the discovery, isolation, and identification of glycoproteins are becoming increasingly important. In this study, a Con A‐magnetic particle conjugate‐based method was utilized to selectively isolate the glycoproteins and their glycomes from the healthy donor and hepatocellular carcinoma (HCC) case sera. The isolated glycoproteins and their N‐linked glycans were identified by LC‐ESI‐MS/MS and MALDI‐TOF/TOF‐MS, respectively. A total of 93 glycoproteins from the healthy donors and 85 glycoproteins from the HCC cases were identified. There were 34 different glycoproteins shown between the healthy donors (21/34) and the HCC cases (13/34). Twenty‐eight glycans from the healthy donors and 30 glycans from the HCC cases were detected and there were 22 different glycans shown between the healthy donors (10/22) and HCC cases (12/22). Among these glycoproteins, 50 were known to be N‐linked glycoproteins and three novel glycopeptides from two predicted potential glycoproteins were discovered. Moreover, lectin blotting, Western blotting and lectin/glyco‐antibody microarrays were applied to definitely elucidate the change of selective protein expressions and their glycosylation levels, the results indicated that the differences of the identified glycoproteins between the healthy donors and HCC cases were caused by the change of both protein expression and their glycosylation levels.     

Fermentation Monitoring and Control: A Perspective

Abstract

One of the fastest growing fields in the pharmaceutical industry is the market for therapeutic glycoproteins. Today, these molecules play a major role in the treatment of various diseases, and include several protein classes, i.e., clotting factors, hormones, cytokines, antisera, enzymes, enzyme inhibitors, Ig-Fc-Fusion proteins, and monoclonal antibodies. Optimal glycosylation is critical for therapeutic glycoproteins, as glycans can influence their yield, immunogenicity and efficacy, which impact the costs and success of such treatments. While several mammalian cell expression systems currently used can produce therapeutic glycoproteins that are mostly decorated with human-like glycans, they can differ from human glycans by presenting two structures at the terminal and therefore most exposed position. First, natural human N-glycans are lacking the terminal Gal? 1–3Gal (alpha-Gal) modification;     

Region-specific upregulation of HNK-1 glycan in the PRMT1-deficient brain

BackgroundBrains express structurally unique glycans, including human natural killer-1 (HNK-1), which participate in development and high-order functions. However, the regulatory mechanisms of expression of these brain-specific glycans are largely unknown. We examined whether arginine methylation, another type of protein modification essential for neural development, impacts the expression of various glycans in the developing brain.MethodsWe analyzed several types of glycans, including the HNK-1 epitope, in the cerebellum and cerebral cortex from mice with nervous system-specific knockout of protein arginine methyltransferase 1 (PRMT1). We also analyzed the expression levels of glycosyltransferases responsible for HNK-1 and of HNK-1 carrier glycoproteins by quantitative RT-PCR and western blotting.ResultsAmong several glycans, expression of HNK-1 glycan was strikingly upregulated in the PRMT1-deficient cerebellum. Furthermore, such upregulation was found in the cerebellum but not in the cerebral cortex. Regarding the mechanisms, we demonstrated that the mRNA level and activity of the responsible glycosyltransferase (B3gat1) were elevated in the knockout cerebellum. We also showed that the expression of HNK-1 carrier glycoproteins such as neural cell adhesion molecule (NCAM), L1 and AMPA receptor subunit GluA2 were also increased in the PRMT1-deficient cerebellum.ConclusionsLoss of arginine methylation leads to an increase in HNK-1 glycan in the developing cerebellum but not in the cerebral cortex via upregulation of the biosynthetic enzyme and carrier glycoproteins.General significancePRMT1 is a novel regulator of HNK-1 glycan production in the cerebellum. Mechanisms involving crosstalk between glycosylation and arginine methylation are suggested to occur.     

Characterization of the human submandibular/sublingual saliva glycoproteome using lectin affinity chromatography coupled to multidimensional protein identification technology

In-depth analysis of the salivary proteome is fundamental to understanding the functions of salivary proteins in the oral cavity and to reveal disease biomarkers involved in different pathophysiological conditions, with the ultimate goal of improving patient diagnosis and prognosis. Submandibular and sublingual glands contribute saliva rich in glycoproteins to the total saliva output, making them valuable sources for glycoproteomic analysis. Lectin-affinity chromatography coupled to mass spectrometry-based shotgun proteomics was used to explore the submandibular/sublingual (SM/SL) saliva glycoproteome. A total of 262 N- and O-linked glycoproteins were identified by multidimensional protein identification technology (MudPIT). Only 38 were previously described in SM and SL salivas from the human salivary N-linked glycoproteome, while 224 were unique. Further comparison analysis with SM/SL saliva of the human saliva proteome, revealed 125 glycoproteins not formerly reported in this secretion. KEGG pathway analyses demonstrated that many of these glycoproteins are involved in processes such as complement and coagulation cascades, cell communication, glycosphingolipid biosynthesis neo-lactoseries, O-glycan biosynthesis, glycan structures-biosynthesis 2, starch and sucrose metabolism, peptidoglycan biosynthesis or others pathways. In summary, lectin-affinity chromatography coupled to MudPIT mass spectrometry identified many novel glycoproteins in SM/SL saliva. These new additions to the salivary proteome may prove to be a critical step for providing reliable biomarkers in the diagnosis of a myriad of oral and systemic diseases.     

Comparison of N-linked Glycoproteins in Human Whole Saliva, Parotid, Submandibular, and Sublingual Glandular Secretions Identified using Hydrazide Chemistry and Mass Spectrometry

INTRODUCTION: Saliva is a body fluid that holds promise for use as a diagnostic fluid for detecting diseases. Salivary proteins are known to be heavily glycosylated and are known to play functional roles in the oral cavity. We identified N-linked glycoproteins in human whole saliva, as well as the N-glycoproteins in parotid, submandibular, and sublingual glandular fluids. MATERIALS AND METHODS: We employed hydrazide chemistry to affinity enrich for N-linked glycoproteins and glycopeptides. PNGase F releases the N-peptides/proteins from the agarose-hydrazide resin, and liquid chromatography-tandem mass spectrometry was used to identify the salivary N-glycoproteins. RESULTS: A total of 156 formerly N-glycosylated peptides representing 77 unique N-glycoproteins were identified in salivary fluids. The total number of N-glycoproteins identified in the individual fluids was: 62, 34, 44, and 53 in whole saliva, parotid fluid, submandibular fluid, and sublingual fluid, respectively. The majority of the N-glycoproteins were annotated as extracellular proteins (40%), and several of the N-glycoproteins were annotated as membrane proteins (14%). A number of glycoproteins were differentially found in submandibular and sublingual glandular secretions. CONCLUSIONS: Mapping the N-glycoproteome of parotid, submandibular, and sublingual saliva is important for a thorough understanding of biological processes occurring in the oral cavity and to realize the role of saliva in the overall health of human individuals. Moreover, identifying glycoproteins in saliva may also be valuable for future disease biomarker studies.