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

Abstract

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.

Introduction

Glycosylation is a posttranslational modification that plays an important role in many cellular processes such as protein conformation, folding, transport, targeting, and stability [1]. Cell growth, differentiation, cell–cell communication, cellular trafficking, immune response, microbial pathogenesis, and other biological processes are influenced by glycosylation [2, 3]. Protein glycosylation is often found to vary with disease progression [4–6]. It is not surprising to note that a significant number of known and Food and Drug Administration-approved biomarkers for diseases are glycoproteins [7], including CA 15-3 [8] and c-erb-2 [9] for breast cancer, prostrate-specific antigen [10] for prostrate cancer, CA125 [11] for ovarian cancer, and carcinoembryonic antigen for ovarian, prostrate, and colon cancers [12]. Glycoproteins represent an interesting subproteome that can be mined to improve the chances for discovering putative robust, specific, and selective biomarkers for diseases.

Biofluids such as serum/plasma, saliva, and urine are excellent starting points for such biomarker discovery studies. Human saliva is gaining popularity as a body fluid for diagnostic purposes. Its ready availability, ease of collection, and ease of storage makes it a potential ideal candidate for use as a diagnostic medium. Whole saliva has contributions from three primary salivary glands: the parotid (PA), submandibular (SM), and sublingual (SL) glands, in addition to other minor contributions (e.g., from numerous minor glands in the lip, cheek, tongue, and palate, microbes, epithelial cells, nasal, and bronchial secretions, and serum products).

Many types of posttranslational modifications have been observed on salivary proteins [13, 14]. Some of the common modifications include glycosylation, phosphorylation, sulfation, acylation, deamidation, and proteolysis. Glycosylation is a posttranslational modification that has important functions in the oral cavity such as lubrication and protection of oral cavity and teeth from oral pathogens, chemicals, and mechanical wear and tear. Some of the salivary glycoproteins involved in lubrication include proline-rich glycoprotein and mucins. Proline-rich glycoproteins are primarily secreted by the PA glands and are N-glycosylated. The oligosaccharides make up to 50% of their weight and are responsible for their lubricating properties [15]. Mucins are also heavily O-glycosylated, which explains their viscous nature and their profound role in oral lubrication [16, 17]. The sugars also help mucins bind to surfaces in the mouth to protect them from chemicals, wear and tear, and microbes [18]. Other functions of salivary glycoproteins include binding to oral pathogens and eliminating them from the oral cavity; proline-rich glycoproteins, mucins, and salivary agglutinins have an especially important part in these functions. Proline-rich glycoproteins bind Fusobacterium nucleatum that causes periodontal diseases and may be responsible for its clearance from the mouth [19]. Mucins also bind and aggregate bacteria such as Heliobacter pylori and flush them from the oral cavity [20]. Salivary agglutinins efficiently adhere to and help rid the oral cavity of Streptococcus mutans [21]. In addition, agglutinins have shown an influenza A-neutralizing effect [22]. After secretion of saliva from the salivary glands into the mouth, glycoproteins may undergo deglycosylation. This might have interesting consequences for oral pathogens and the function of glycoproteins. Many bacteria are known to deglycosylate glycoproteins such as mucins and utilize the released sugars for their growth [23]. Some Streptococcus species secrete neuraminidases that cleave the terminal sialic acid from mucins. This affects the ability of mucins to bind and aggregate bacteria such as Streptococcus sanguis [24]. The removal of sialic acid may also help bacteria bind to sugars on glycoproteins and colonize in the oral environment [14].

Mapping the salivary glycoproteome will be an important step toward using saliva for disease diagnosis. Whole saliva is far easier to collect compared to ductal saliva, and whole saliva will likely have more applications for disease biomarker studies compared to PA, SM, and SL saliva. However, mapping the N-glycoproteome of PA, SM, and SL 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.

Several methods for global glycoprotein profiling of biofluids have been explored to date, and many of the same techniques have also been applied to enrich for disease biomarkers. Some of the common methodologies tested include lectin affinity purification [25], hydrazide-based chemical derivatization for glycoprotein capture, and hydrophilic affinity capture methods. Lectins are a class of proteins found in plants, bacteria, fungi, and animals that bind to specific oligosaccharides on glycoproteins [26]. Whereas the use of a single lectin can help to enrich for a small group of glycoproteins, the use of multiple lectins increases the chances for isolating a broad spectrum of glycoproteins. This method, termed multilectin affinity chromatography, has been used to map glycoproteins in the plasma and serum [27–29]. To enrich for glycoproteins to which lectins do not have a strong specificity, the use of serial lectin affinity chromatography (SLAC) has been reported [30]. Because there are few reported lectins with sufficient selectivity for O-linked glycoproteins, SLAC has been used to isolate and identify O-linked glycopeptides in human serum [30]. The use of a broad-spectrum lectin such as concanavilin A was used to deplete N-linked glycoproteins and to enrich for O-linked glycoproteins. Jacalin is a lectin that has affinity for the GalNAc core of O-linked glycans but also for high mannose-type sugars found in N-linked glycoproteins. The subsequent application of the N-glycoprotein-depleted sample to a Jacalin column improved recovery and identification of O-linked glycoproteins [30]. Lectin affinity chromatography has also been employed to search for disease biomarkers in human plasma and serum [31, 32].

Phenyl boronic acid is considered a pseudolectin because it covalently attaches to cis-diol containing groups on the sugars of glycoproteins. This bond is fairly stable under alkaline conditions. Thus, boronic acids can be utilized to select for a broader group of glycoproteins including N-, O-, and C-linked glycoproteins. Lectins and boronic acid can be combined in a single experiment to enrich for glycoproteins [33]. Sparbier and coworkers used a combination of lectin and boronic acid magnetic beads to affinity select glycoproteins from human serum [34].

Hydrophilic affinity-based capture methods have been extended to isolate glycoproteins from complex mixtures. Because sugars are very hydrophilic, glycoproteins lend themselves well to being captured onto a hydrophilic stationary phase. Several hydrophilic materials have been tested in the past. Some of them include carbohydrate gel matrices such as sepharose and cellulose and hydrophilic interaction chromatography (HILIC) columns. Cellulose and sepharose matrices were successfully employed to isolated glycoproteins from human serum [35]. HILIC using a resin containing a polyhydroxyethyl aspartamide group has been used to separate sialylated glycopeptides from recombinant human interferon-γ [36]. Hagglund and coworkers used a resin with a covalently bound neutral, zwitterionic sulfobetaine functional group to perform zwitterion chromatography–HILIC (ZIC-HILIC). Glycopeptides were separated via ZIC-HILIC from nonglycopeptides in trypsin digest mixtures and were then partially deglycosylated to identify glycoproteins in human plasma [37, 38].

Larsen and coworkers published an elegant method to isolate sialic acid-containing glycoproteins in plasma and whole saliva. Their method employs titanium dioxide, which has an affinity for acidic groups. TiO₂ has been used commonly to purify and measure phosphoproteins [39]. However, it also binds to sialic acid-containing glycoproteins. After removing phospho-groups on phosphopeptides by alkaline phosphatase treatment, the peptide mixture was passed through the TiO₂ column to isolate sialated glycopeptides, and the glycopeptides were analyzed subsequently by mass spectrometry (MS). Using this method, sialated proteins in plasma and whole saliva were identified [40].

Zhang and coworkers published a landmark paper describing the isolation of formerly N-linked glycopeptides from complex mixtures using hydrazide chemistry [41]. This method has gained popularity over the past few years. Sugars on glycoproteins are first oxidized and immobilized onto an agarose–hydrazide resin. The resin is then washed to remove nonspecifically bound proteins. The proteins bound to the resin are digested with trypsin, and tryptic peptides not covalently bound to the column are removed by extensive washing. The bound tryptic N-glycopeptides are then released using the enzyme PNGase F, and the formerly N-glycosylated peptides are analyzed by MS. The identification of the formerly N-glycosylated peptide is confirmed by the presence of an N–X–S/T motif on the peptide sequence (where X denotes any amino acid residue except proline) and the conversion of asparagines at the site for glycosylation to aspartic acid resulting in a mass increase of +1 Da. This method has since been widely applied toward the study of the N-glycoproteome of various human body fluids including plasma [42], serum [43], whole saliva [44], and cerebrospinal fluid [45]. In 2007, two independent groups published slightly modified versions of the original method described by Zhang et al. [41]. The similarity between the two methods was that the proteins initially were digested to tryptic peptides before being coupled onto the agarose–hydrazide resin. In the method published by Sun et al., excess sodium periodate, used to oxidize sugars on glycopeptides, was quenched with sodium sulfite. The method was used to identify unique proteins from microsomal fractions of the cisplatin-resistant ovarian cancer cell lines [46]. Alternately, Tian et al. removed remaining sodium periodate by desalting the oxidized glycopeptides using a C18 column [47]. This technique was successfully used to isolate N-linked glycopeptides from mouse plasma including some proteins that are found in low abundance in human plasma [48]. There are also differences in the wash step to remove nonspecifically bound peptides from the agarose–hydrazide resin, but the basic outline of the two procedures remains similar.

Our group previously reported, using the method of Zhang et al. [41], a global N-glycoproteome analysis of whole-saliva proteins using the hydrazide capture technique and MS, identifying 84 formerly N-glycosylated peptides from 45 glycoproteins [44]. Larsen et al. used the TiO₂ enrichment strategy to isolate the whole salivary sialome and identified 97 sites of N-linked glycosylation [40]. In this paper, we extend the salivary glycoprotein catalogue using the modified hydrazide capture method of Sun et al. [46], i.e., the capture of tryptic N-glycopeptides and MS measurement of formerly N-linked glycopeptides. In addition, we compare for the first time the N-linked glycoproteins identified in PA, SM, and SL saliva.

Materials and Methods

Chemicals and Reagents

The chemicals were mostly purchased from Sigma (St. Louis, MO, USA), unless stated otherwise. Affigel Hz hydrazide gel, coupling buffer, and dithiothreitol (DTT) were obtained from Bio-Rad (Hercules, CA, USA). Tris-(2-carboxyethyl)-phosphine (TCEP) and trifluoroacetic acid (TFA) were obtained from Pierce (Rockford, IL, USA). Glycerol-free PNGase F was obtained from New England Biolabs (Ipswich, MA, USA). Sequencing-grade trypsin was procured from Promega (Madison, WI, USA).

Saliva Collection

Whole saliva was collected from healthy nonsmoking adults in the morning, at least 2 h after the last intake of food. The mouth was rinsed with water immediately before the collection. Whole saliva was collected and placed on ice. Protease cocktail inhibitor (1 µL/mL of whole saliva) was added to saliva immediately after collection to minimize protein degradation. Whole saliva was then centrifuged at 12,000 rpm at 4°C for 10 min. The supernate was collected and stored at −80°C. The pellet was saved for future analysis.

For collecting PA, SM, and SL saliva, 10 adult subjects of various ethnic and racial backgrounds, ranging in age from 22 to 30 years, were recruited. Saliva collection took place on a monthly basis and was performed between the hours of 9 and 11 a.m. Stimulated PA, SM, and SL saliva were collected by the repeated application of an aqueous citric acid solution (2%). PA saliva was obtained as the ductal secretion by using a cup-like device [49]. Separate SM and SL secretions were acquired by using a saliva collector, described by Wolff and coworkers, that was fitted with a sterile 100-µL-pipette tip [50]. Collection volumes over a 10-min period ranged from 500 to 2,000 µL for PA saliva, 50 to 100 µL for SL saliva, and 100 to 500 µL for SM saliva. PA, SM, and SL saliva from five subjects were pooled before carrying out the experiments.

Solution Isoelectric Focusing Fractionation

The procedure for solution isoelectric focusing (IEF) fractionation has been described previously [44]. Briefly, proteins in whole saliva were precipitated by mixing with four times the volume of cold ethanol and incubating at −20°C overnight. The mixture was centrifuged at 13,000 rpm for 15 min at 4°C. The pellet was resuspended in Zoom 2D protein solubilizer (Invitrogen, Carlsbad, CA, USA), protease inhibitor (Roche Diagnostic, Indianapolis, IN, USA), Tris base, DTT, and water and sonicated on ice. The pH of the solution was adjusted to pH 8.5–8.7 with 1 M Tris base and then incubated for 15 min at room temperature with shaking. Proteins were alkylated with 99% dimethylacrylamide (DMA) at room temperature for 30 min, as suggested by the protocols from Invitrogen. To quench excess DMA, DTT was added and incubated at room temperature for 5 min. After centrifuging the sample for 30 min at 13,400 rpm at 4°C, the supernate was collected. The protein concentration was determined by the noninterfering protein assay (Geno Technology, St. Louis, MO, USA). The concentration was measured to be approximately 1.5 mg/mL.

The solution of proteins in the Zoom-2D solubilizer (1.5 mg/mL, 400 µL) was diluted to a final concentration of 0.6 mg/mL in diluted buffer (Zoom IEF denaturant, Zoom focusing buffer pH 3–7, Zoom focusing buffer, pH 7–12, and 5 µL 2M DTT). Zoom IEF fractionation was performed in the standard format (pH 3.0 to 10). The diluted sample was loaded into each of the five chambers of the fractionator and subjected to solution-phase IEF. Five fractions (pI 3–4.6, 4.6–5.4, 5.4–6.2, 6.2–7.0, and 7.0–10.0) were obtained after the procedure. Proteins from each fraction were precipitated by mixing with 70% acetone, incubating at −20°C for 3–4 h, and centrifuging at 13,000 rpm for 30 min.

Glycoprotein Enrichment

Proteins from saliva that were not previously pI fractionated were isolated by precipitation using ethanol. Proteins from Zoom IEF-separated fractions were obtained by acetone precipitation. The salivary proteins isolated by either method were resuspended in coupling buffer (100 mM sodium acetate, 150 mM sodium chloride, pH 5.5). Sodium periodate was added to a final concentration of 15 mM. The solution was incubated in the dark at room temperature for 1 h. Glycerol was added to a final concentration of 20 mM to quench any excess sodium periodate remaining in the solution. The mixture was incubated for 15 min with mixing at room temperature. To remove remaining sodium periodate, the solution was dialyzed using a 3.5-kDa dialysis cassette against 1× coupling buffer at 4°C overnight. The hydrazide resin was equilibrated by washing with 3 vol of water and 6 vol of coupling buffer. The proteins were added to the resin and coupled overnight by incubating overnight at room temperature with shaking. The resin was then allowed to settle, and the supernate containing uncoupled nonglycoproteins was discarded. The resin was washed six times with urea buffer A containing 8 M urea, 200 mM Tris, 0.05% sodium dodecyl sulfate, and 5 mM ethylenediamine tetraacetic acid (pH 8.3). The proteins on the resin were reduced with a solution of 10 mM TCEP in urea buffer A. The reduced proteins were alkylated with 50 mM iodoacetamide in urea buffer. The resin was then washed six times with urea buffer B (1 M urea, 25 mM Tris, pH 8.3). The resin was resuspended in urea buffer B. Trypsin was added to the solution, and the proteins attached to the resin were digested at 37°C overnight with shaking. The nonglycopeptides released by trypsin digestion were removed by washing three times with 1.5 M NaCl, 80% acetonitrile (ACN)/0.1% TFA, methanol, and water and six times with 100 mM ammonium bicarbonate. The resin was resuspended in 100 mM ammonium bicarbonate. The N-linked glycopeptides were released by adding PNGase to the resin and incubating at 37°C overnight. The resin was washed twice with 80% ACN solution. The washes were combined, and the released glycopeptides were dried by vacuum centrifugation. The peptides were then resuspended in 0.1% formic acid (FA) and analyzed by one-dimensional (1D) liquid chromatography (LC)–MS/MS.

Glycopeptide Enrichment

The method used followed that described by Sun et al. [46]. Proteins from whole saliva, PA, SM, and SL saliva were precipitated using cold ethanol. The pellets were resuspended in 50 mM ammonium bicarbonate buffer (pH 7.8) with sonication. TCEP was added to a final concentration of 10 mM at room temperature to reduce disulfide bonds (TCEP is preferred over DTT in this case because elevated temperatures required by DTT–disulfide reduction often causes protein precipitation from salivary fluids). The solution was incubated at room temperature for 30 min with shaking. Iodoacetamide (50 mM) was added to alkylate the reduced cysteines, and the mixture was incubated at room temperature for 30 min with shaking. Afterwards, DTT (25 mM) was added to the solution to quench any remaining unreacted iodoacetamide. The mixture was incubated at room temperature for 30 min. Trypsin was added (1:50 by weight) to digest the proteins in solution, and the solution was incubated at 37°C overnight. The tryptic peptide mixture was then acidified with FA to bring the pH below 4.0. The tryptic peptides were desalted using a C18 Sep-Pak reverse-phase (RP) column (Waters, Milford, MA, USA). Peptides were then dried and resuspended in coupling buffer (100 mM sodium acetate, 150 mM sodium chloride, pH 5.5). Sodium periodate was added to a final concentration of 10 mM to oxidize cis-diol groups on sugars. The mixture was incubated in the dark at room temperature for 1 h. Any remaining unreacted sodium periodate was quenched with sodium sulfite to a final concentration of 20 mM. The solution was incubated at room temperature for 10 min. The mixture was then added to agarose–hydrazide resin to couple the peptides to the resin, and the coupling reaction was allowed to proceed overnight at room temperature. The agarose-hydrazide resin was transferred to a Handee minispin column (Pierce, Rockford, IL, USA) and washed with 1.5 M NaCl, 80% ACN, methanol, water, and freshly prepared 100 mM ammonium bicarbonate solution. The resin was incubated overnight at 37°C with PNGase F in 100 mM ammonium bicarbonate to release formerly N-glycosylated peptides. Afterwards, the resin was washed twice with 80% ACN solution. The washes were combined, and the released glycopeptides were dried in the vacuum centrifuge. The formerly N-glycosylated peptides were then resuspended in 1% FA and analyzed by 1D- and two-dimensional (2D) LC-MS/MS.

Liquid Chromatography–Tandem Mass Spectrometry

LC-MS/MS and 2D LC-MS/MS were performed on a QSTAR XL (QqTOF) mass spectrometer (Applied Biosystems, Foster City, CA, USA) equipped with a nanoelectrospray (Protana, Odense, Denmark) interface and an LC Packings/Dionex (Sunnyvale, CA, USA) nano-LC system.

For 1D LC-MS/MS, the nano-LC was equipped with a set of homemade precolumns (75 µm× 10 mm) and a column (75 µm× 150 mm) packed with Jupiter Proteo C12 resins (particle size 4 µm, Phenomenex, Torrance, CA, USA). The peptides were dried and redissolved in 0.1% FA solution. For each LC-MS/MS run, typically, 6 µL sample solution was loaded onto the precolumn. The precolumn was washed with the loading solvent containing 0.1% FA for 4 min before the sample was injected onto the LC column. The eluents used for the LC were water containing 0.1% FA (A) and 95% ACN/water containing 0.1% FA (B). The flow was 200 nL/min. The following analytical LC gradient was used for analyzing the formerly N-glycosylated peptides obtained by the glycopeptide enrichment method: 3–21% B for 36 min, 21–35% B for 14 min, and 36–80% B for 4 min and held at 80% B for 10 min. The column was equilibrated with 3% B for 16 min before the next run. The gradient used for analyzing formerly N-glycosylated peptides obtained by the glycoprotein enrichment method was 3–35% B for 72 min and 35–80% B for 18 min and maintained at 80% B for 9 min.

For the online 2D LC-MS/MS, 20 µL of sample solution was loaded onto a strong cation exchange (SCX) precolumn (Luna SCX resin, particle size 5 µm, 150 µm× 5 mm, Phenomenex) before transfer to a RP precolumn (Jupiter Proteo C12 resin, particle size 4 µm, 150 µm× 5 mm, Phenomenex) and RP analytical column (Jupiter Proteo C12 resin, particle size 4 µm, 75 µm× 150 mm, Phenomenex). Seven concentrations of ammonium acetate solutions (50, 100, 200, 400, 600, 1,000, and 2,000 mM) were injected (4 µL) onto the SCX precolumn for the step gradient elution of peptides.

The RP precolumn was used to preconcentrate and desalt each peptide fraction eluted from the SCX column prior to nano-RP LC separation. SCX fractions were loaded onto the RP precolumn with the following gradient: 3% B for 6 s, 6–24% B for 18 min, 24–36% B for 6 min, and 36–80% B for 2 min and maintained at 80% B for 8 min. The column was equilibrated with 3% B for 15 min prior to the next run. SCX fractions were separated on the analytical RP column (200 nL/min) with the following gradient: 3% B for 5 min, 3–6% B for 6 s, 6–24% B for 18 min, 24–36% B for 6 min, and 36–80% B for 2 min and held at 80% B for 8 min.

For online MS/MS analyses, a Proxeon nanobore stainless steel online emitter (inner diameter=30 µm) was used for electrospraying with the voltage set to 1,900 V. Peptide product ion mass spectra were recorded during LC-MS/MS by information-dependent analysis on the QSTAR XL mass spectrometer. Argon was employed as the collision gas. Collision energies for maximum fragmentation were automatically calculated using empirical parameters based on the charge and mass-to-charge ratio of the precursor peptide.

Protein identification was accomplished utilizing the Mascot database search engine (Matrix Science, London, UK). For search the sequence databases, the following variable modifications were set: carbamidomethylation of cysteines, oxidization of methionines and enzyme-catalyzed conversion of asparagines to aspartic acid at the site of carbohydrate attachment on asparagines, conversion of N-terminal glutamate and aspartate to pyro-Glu, and cyclization of N-terminal cysteine. DMA modification of cysteine was set as one of the parameters in experiments where Zoom IEF fractionation had been done prior to glycoprotein pulldown. Searches for peptides obtained by the glycoprotein enrichment method were performed against the Human IPI database. For the searches, one missed tryptic cleavage was tolerated, and the peptide and MS/MS mass tolerance was set as 0.3 Da. A Mascot score of greater than 20 was considered a significant match. All matching peptide MS/MS spectra were manually examined to verify the accuracy of the identification. Positive protein identification was based on standard Mascot criteria for statistical analysis of the LC-MS/MS data. For samples obtained by the glycopeptide enrichment method, searches were done against the Human IPI database (version 3.39) and its reverse decoy database. Only peptides above Mascot’s homology or identity threshold were considered. The validity of the formerly N-glycosylated peptides were confirmed by the presence of a consensus N–X–(S/T) where X is any amino acid except proline and the conversion of asparagines to aspartic acid at the site of former N-glycosylation resulting in a mass change of +1 Da. With MS/MS tolerances set to 0.3 Da, a 1-Da difference caused by deamidation of Asn to Asp is readily detected from measurements performed on the QTOF mass spectrometer.

Results

Isolation of N-linked Glycoproteins

In our previous study on salivary N-linked glycoproteins [44], we used the agarose–hydrazide glycoprotein pulldown technique of Zhang et al. [41] to characterize human whole saliva (we will henceforth refer to this technique as the “Zhang method”). Proteins from whole saliva were isolated in two different ways. In the first method, proteins in whole saliva were precipitated with ethanol, and glycoprotein pulldown proceeded thereafter. The second method involved the preseparation of proteins into five different pI fractions of 3–4.6, 4.6–5.4, 5.4–6.2, 6.2–7, and 7–10, by solution IEF fractionation. The proteins in each pI fraction were then precipitated using acetone. The steps employed for glycoprotein pulldown in our previous study using the Zhang method were: (a) resuspending whole salivary proteins in the coupling buffer, (b) oxidizing saccharides on the glycoproteins with sodium periodate, (c) quenching excess sodium periodate with glycerol and removing any remaining sodium periodate by dialyzing against coupling buffer, (d) coupling glycoproteins to the agarose–hydrazide resin, (e) washing nonglycosylated proteins, (f) digesting proteins attached to the hydrazide resin with trypsin, (g) washing away the nonglycopeptides, (h) releasing the formerly N-glycosylated proteins from the hydrazide resin with PNGase F, and (i) identifying the formerly N-glycopeptides by 1D LC-MS/MS (Fig. 1a). A total of eight repetitions were performed for the glycoprotein pulldown experiments where saliva proteins were previously precipitated by ethanol precipitation. Five repetitions of the IEF fractionation followed by glycoprotein pulldown were performed.

Fig. 1.

Steps involved in the isolation of formerly N-glycosylated peptides using the a Zhang method and b the Sun method

In this current study, we applied a modified version of the glycoprotein pulldown method [46] to extend our catalogue of whole-saliva N-linked glycoproteins and developed a new catalogue of N-glycoproteins from segregated PA, SM, and SL fluids (we will refer to this modified technique as the “Sun method” in this paper). The new method entails proteolytic digestion of proteins prior to coupling to the agarose–hydrazide resin. The other difference lies in that after the oxidation of sugars, any unreacted sodium periodate is quenched with sodium sulfite. This obviates the need to remove sodium periodate by dialysis or using a desalting column as performed in the previous study. In this current study, the salivary proteins were precipitated using ethanol. Thus, the steps to isolate N-glycoproteins are: (a) resuspending salivary proteins in ammonium bicarbonate buffer, (b) digesting proteins with trypsin, (c) desalting the tryptic peptides using a C18 column, (d) oxidizing sugars on glycopeptides with sodium periodate, (e) quenching any remaining sodium periodate with sodium sulfite, (f) coupling glycopeptides onto the agarose–hydrazide column, (g) washing away nonglycopeptides, (h) releasing formerly N-linked glycopeptides with PNGase F, and (i) identifying formerly N-linked glycopeptides using 1D and 2D LC-MS/MS (Fig. 1b). A total of eight 1D LC-MS/MS runs were carried out. Some of the samples were combined, and four additional 2D LC-MS/MS experiments were performed. Data obtained from both 1D and 2D LC-MS/MS runs were combined. In both methods, the formerly N-glycosylated peptide identifications were validated by the presence of the consensus N–X–(S/T) motif and conversion of asparagines to aspartic acid at the site of N-glycosylation. We applied the Sun method to identify N-glycoproteins in whole saliva, PA, SM, and SL saliva.

Identification of N-Glycoproteins in Whole Saliva

In our previous study on N-linked glycoproteins in human whole saliva, we reported 84 formerly N-linked glycopeptides from 44 unique N-glycoproteins (Table 1) [44]. Using the newer Sun method, we identified 80 formerly N-glycosylated peptides from 46 unique N-linked glycoproteins (Table 1). However, comparing our previously identified N-glycoproteins using the Zhang method and new data set obtained by the Sun method, we found that 42 formerly N-glycosylated peptides from 28 N-glycoproteins were identified by both methods, 42 formerly N-glycosylated peptides from 16 N-glycoproteins were identified by the Zhang method uniquely, and 38 formerly N-glycopeptides from 18 N-glycoproteins were identified uniquely by the Sun method. Combining all of the N-glycoproteins identified by both methods, we have thus far identified 122 formerly N-glycosylated peptides from 62 unique N-glycoproteins (Fig. 2a,b). Figure 2c shows the LC-MS/MS spectrum of a formerly N-glycosylated peptide GDQLILNLNN*ISSDR (where the asterisk denotes the site of N-glycosylation) from Isoform 1 of long palate, lung, and nasal epithelium carcinoma-associated protein 1 identified in whole saliva using the Sun method, whereas this formerly N-glycopeptide was not detected in PA, SM, or SL saliva (vide infra).

Table 1.

Comparison of N-linked glycoproteins identified in whole saliva using the Zhang method and the Sun method

IPI Accession number	Protein name	MW (Da)	Formerly N-glycosylated peptide sequencea	Formerly N-glycosylated peptides identified		Nonformerly N-glycosylated peptides identified
				Zhang methodb	Sun methodb	Zhang method	Sun method
IPI00002818	Splice isoform 1 of kallikrein 11	27,448	CAN*ITIIEHQK	No	Yes (35)	–	0
IPI00003351	Extracellular matrix protein 1	60,635	HIPGLIHN*MTAR	No	Yes (43)	–	0
IPI00004573	Polymeric-immunoglobulin receptor	83,232	VPGN*VTAVLGETLK	Yes (101)	Yes (113)	18	3
			WNN*TGCQALPSQDEGPSK	Yes (72)	Yes (62)
			IIEGEPNLKVPGN*VTAVLGETLK	Yes (49)	Yes (112)
			LSLLEEPGN*GTFTVILNQLTSR	Yes (60)	Yes (99)
			ANLTNFPEN*GTFVVNIAQLSQDDSGR	Yes (70)	Yes (108)
			QIGLYPVLVIDSSGYVNPN*YTGR	Yes (95)	Yes (112)
			VPGN*VTAVLGETLKVPCHFPCK	No	Yes (50)
IPI00007244	Myeloperoxidase	83,815	SCPACPGSN*ITIR	Yes (46)	No	2	–
			SYN*DSVDPR	Yes (41)	No
IPI00011229	Cathepsin D	44,524	GSLSYLN*VTR	No	Yes (49)	–	0
IPI00855918	Mucin 5, subtype B, tracheobronchial	590,122	VVLLDPKPVAN*VTCVNK	Yes (60)	Yes (80)	37	0
			AQGLVLEASN*GSVLINGQR	Yes (118)	Yes (120)
			GN*CTYVLMR	Yes (32)	Yes (39)
			AFGQFFSPGEVIYN*K	No	Yes (72)
			AFGQFFSPGEVIYN*KTDR	Yes (49)	Yes (72)
			DIECQAESFPN*WTLAQVGQK	Yes (82)	Yes (86)
			QVNETWTLEN*CTVAR	Yes (47)	Yes (67)
			LDGPTEQCPDPLPLPAGN*CTDEEGICHR	Yes (65)	Yes (30)
			FGN*LSLYLDNHYCTASATAAAAR	Yes (49)	No
			LPYSLFHN*NTEGQCGTCTNNQR	Yes (32)	No
IPI00012887	Cathepsin L	37,540	YSVAN*DTGFVDIPK	No	Yes (57)	–	0
IPI00013972	Carcinoembryonic antigen-related cell adhesion molecule 8	38,130	LFIPN*ITTK	Yes (47)	No	0	–
IPI00019943	Afamin	69,024	DIENFN*STQK	Yes (41)	No	0	–
IPI00020091	Alpha-1-acid glycoprotein 2	23,588	QNQCFYN*SSYLNVQR	Yes (90)	No	0	–
IPI00020487	Extracellular glycoprotein lacritin	14,237	QFIEN*GSEFAQK	Yes (32)	No	1	–
IPI00020986	Lumican	38,405	LHINHNN*LTESVGPLPK	No	Yes (59)	–	0
IPI00021891	Splice Isoform 1 of Fibrinogen gamma chain	51,479	DLQSLEDILHQVEN*K	No	Yes (58)	–	0
			VDKDLQSLEDILHQVEN*K	No	Yes (70)
IPI00022395	Complement component C9	63,133	AVN*ITSENLIDDVVSLIR	No	Yes (56)	–	0
IPI00022417	Leucine-rich alpha-2-glycoprotein	38,944	KLPPGLLAN*FTLLR	Yes (62)	Yes (72)	1	0
IPI00022429	Alpha-1-acid glycoprotein 1	24,777	QDQCIYN*TTYLNVQR	Yes (105)	No	0	–
IPI00022431	Alpha-2-HS-glycoprotein	39,300	AALAAFNAQNN*GSNFQLEEISR	Yes (64)	Yes (26)	1	–
			VCQDCPLLAPLN*DTR	Yes (40)	No
IPI00022463	Serotransferrin	77,000	CGLVPVLAENYN*K	No	Yes (69)	9	0
			QQQHLFGSN*VTDCSGNFCLFR	Yes (62)	No
IPI00022488	Hemopexin	51,643	ALPQPQN*VTSLLGCTH	Yes (56)	Yes (58)	1	0
			SWPAVGN*CSSALR	Yes (47)	No
IPI00022974	Prolactin inducible protein	16,562	TFYWDFYTN*R	Yes (50)	No	5	–
IPI00023673	Galectin-3 binding protein	65,289	ALGFEN*ATQALGR	Yes (83)	Yes (80)	2	0
			AAIPSALDTN*SSK	Yes (64)	Yes (70)
			GLN*LTEDTYKPR	No	Yes (49)
			EPGSN*VTMSVDAECVPMVR	No	Yes (41)
			TVIRPFYLTN*SSGVD	Yes (57)	No
			YKGLN*LTEDTYKPR	Yes (50)	No
IPI00025023	Lactoperoxidase	80,237	KPSPCEFIN*TTAR	Yes (34)	Yes (53)	8	0
			IVGYLNEEGVLDQN*R	Yes (77)	Yes (89)
			LRN*LSSPLGLMAVNQEVSDHGLPYLPYDSK	No	Yes (51)
IPI00025753	Desmoglein 1	113,644	TGEIN*ITSIVDR	Yes (36)	No	0	–
IPI00025846	Splice isoform 1 of desmocollin-2	99,899	NGIYN*ITVLASDQGGR	No	Yes (88)	1	0
			AN*YTILK	Yes (49)	Yes (48)
			AN*YTILKGNENGNFK	Yes (26)	No
			LKAIN*DTAAR	Yes (31)	No
IPI00027412	Carcinoembryonic antigen-related cell adhesion molecule 6	37,214	LQLSNGN*MTLTLLSVK	Yes (63)	No	0	–
IPI00027486	Carcinoembryonic antigen-related cell adhesion molecule 5	76,748	TLTLFN*VTR	Yes (42)	No	0	–
IPI00031019	Cystatin-related epididymal spermatogenic protein	16,265	KLKPVN*ASNANVK	Yes (43)	No	–	–
IPI00031121	Carboxypeptidase E	53,117	DLQGNPIAN*ATISVEGIDHDVTSAK	No	Yes (69)	–	0
			GN*ETIVNLIHSTR	No	Yes (43)
IPI00031547	Desmoglein 3	107,435	NTGDIN*ITAIVDR	No	Yes (80)	0	0
			LPAVWSITTLN*ATSALLR	Yes (39)	No
			DSTFIVN*K	Yes (45)	No
IPI00032258	Complement C4	192,650	GLN*VTLSSTGR	Yes (59)	Yes (56)	0	0
IPI00032292	Metalloproteinase inhibitor 1	23,156	FVGTPEVN*QTTLYQR	Yes (88)	Yes (101)	2	–
			SHN*RSEEFLIAGK	Yes (29)	No
IPI00060143	Protein FAM3D	24,947	GLNIALVN*GTTGAVLGQK	Yes (56)	Yes (83)	0	0
IPI00166729	Alpha-2-glycoprotein 1, zinc	34,223	DIVEYYNDSN*GSHVLQGR	Yes (91)	Yes (102)	13	0
			FGCEIENN*R	Yes (57)	No
			FGCEIENN*RSSGAFWK	Yes (58)	No
IPI00171411	Golgi membrane protein 1	49,768	AVLVNN*ITTGER	Yes (80)	Yes (68)	1	0
			LQQDVLQFQKN*QTNLER	No	Yes (56)
IPI00178926	Immunoglobulin J chain	18,087	EN*ISDPTSPLR	Yes (55)	Yes (75)	6	0
			IIVPLNNREN*ISDPTSPLR	Yes (56)	Yes (68)
IPI00218460	Isoform 3 of attractin	153,687	IDSTGN*VTNELR	No	Yes (59)	–	0
IPI00242956	Fc fragment of IgG binding protein	571,718	VVTVAALGTN*ISIHKDEIGK	No	Yes (53)	11	1
			VITVQVAN*FTLR	Yes (58)	Yes (50)
			VTVRPGESVMVN*ISAK	No	Yes (54)
			KVTVRPGESVMVN*ISAK	No	Yes (34)
			YLPVN*SSLLTSDCSER	Yes (98)	Yes (46)
IPI00291410	Isoform 1 of long palate, lung, and nasal epithelium carcinoma-associated protein 1	54,878	DHN*ATSILQQLPLLSAMR	No	Yes (87)	–	0
			GDQLILNLNN*ISSDR	No	Yes (87)
IPI00291488	Splice isoform of WAP four-disulfile core domain protein 2	12,984	TGVCPELQADQN*CTQECVSDSECADNLK	Yes (94)	No	0	–
IPI00295105	Carbonic anhydrase VI	35,343	LENSLLDHRN*K	No	Yes (38)	–	0
IPI00296099	Thrombospondin 1	129,330	VVN*STTGPGEHLR	Yes (44)	No	0	–
IPI00296654	Bactericidal permeability-increasing protein-like 1	49,100	LGATPVAMLHTNN*ATLR	Yes (39)	Yes (71)	5	0
			LLAAAN*FTFK	Yes (64)	Yes (66)
			SDDNLLN*TSALGR	Yes (81)	No
IPI00297910	Tumor-associated calcium signal transducer 2	35,687	HRPTAGAFN*HSDLDAELR	No	Yes (34)	–	0
IPI00298082	Calcium-activated chloride channel protein 2	101,283	DSFDDALQVN*TTDLSPK	Yes (38)	No	0	–
IPI00298237	Tripeptidyl-peptidase I	61,708	FLSSSPHLPPSSYFN*ASGR	No	Yes (65)	–	0
IPI00298828	Beta-2-glycoprotein I	38,273	VYKPSAGN*NSLYR	Yes (40)	Yes (74)	0	0
IPI00299547	Neutrophil gelatinase-associated lipocalin	22,774	EDKSYN*VTSVLFR	Yes (28)	No	3
			SYN*VTSVLFR	Yes (55)	No
IPI00299729	Transcobalamin I	48,164	ADEGSLKN*ISIYTK	Yes (50)	Yes (51)	3	0
			MN*DTIFGFTMEER	Yes (32)	Yes (81)
			AQKMN*DTIFGFTMEER	Yes (49)	No
IPI00300786	Alpha-amylase, salivary	57,731	NVVDGQPFTNWYDN*GSNQVAFGR	Yes (108)	Yes (129)	16	9
IPI00304557	Short palate, lung, and nasal epithelium carcinoma associated protein 2	26,995	AEPIDDGKGLN*LSFPVTAN*VTVAGPIIGQIINLK	Yes (55)	No	0	–
			GLNLSFPVTAN*VTVAGPIIGQIINLK	Yes (53)	No
IPI00328960	Similar to carcinoembryonic antigen-related cell adhesion molecule 1	91,568	FVTAGSN*VTLR	No	Yes (31)	–	0
IPI00553177	Alpha-1-antitrypsin	48,208	YLGN*ATAIFFLPDEGK	No	Yes (100)	–	0
IPI00328960	Predicted: hypothetical protein XP_085831	91,568	TPASN*ISTQVSHTK	Yes (44)	No	0	–
IPI00333140	Delta notch-like EGF repeat containing transmembrane	78,438	LVSFEVPQN*TSVK	Yes (43)	Yes (40)	1	–
IPI00335356	IGHM protein	49,526	GLTFQQN*ASSMCVPDQDTAIR	No	Yes (40)	–	0
IPI00374315	Uncharacterized protein C6orf58	37,902	IILN*QTAR	Yes (52)	Yes (5)	1	0
			MGMYKIILN*QTAR	Yes (72)	Yes (63)
IPI00419215	Alpha-2-macroglobulin-like protein 1	78,438	LGHIN*FTISTK	No	Yes (35)	–	0
IPI00641229	Ig alpha-2 chain C region	56,111	LAGKPTHVN*VSVVMAEVDGTCY	Yes (56)	Yes (97)	9	1
			LSLHRPALEDLLLGSEAN*LTCTLTGLR	No	Yes (109)
			TPLTAN*ITK	Yes (57)	No
IPI00400826	Clusterin	57,796	LAN*LTQGEDQYYLR	Yes (64)	Yes (88)	0	0
IPI00418512	DMBT1	259,409	LVNLN*SSYGLCAGR	Yes (95)	Yes (89)	3	0
			QADN*DTIDYSNFLTAAVSGGIIK	Yes (110)	Yes (112)
			CSGN*ESYLWSCPHK	Yes (38)	No
IPI00431645	HP protein	31,362	NLFLN*HSEN*ATAK	Yes (59)	Yes (63)	3	0
			VVLHPN*YSQVDIGLIK	No	Yes (74)
			MVSHHN*LTTGATLINEQWLLTTAK	No	Yes (68)
IPI00168728	FLJ00385 protein (fragment)	56,075	EEQFN*STFR	No	Yes (51)	–	0
IPI00550991	Alpha-1-antichymotrypsin	50,596	FN*LTETSEAEIHQSFQHLLR	No	Yes (52)	–	0

^aN* indicates the site of former N-linked glycosylation

^bMascot score listed in parentheses

Fig. 2.

Comparison of a formerly N-glycosylated peptides and the b N-glycoproteins they represent identified in whole saliva using the Zhang method and the Sun method. c MS/MS mass spectrum of a doubly charged peptide, GDQLILNLNN*ISSDR (m/z 837; asterisk denotes the site of N-glycosylation), from isoform 1 of long palate, lung, and nasal epithelium carcinoma (PLUNC)-associated protein 1 measured from whole saliva

The seemingly poor overlap in protein identification between the two methods could be attributed to several factors. The method of collection of whole saliva has been the same in both studies. However, the glycoprotein pulldown experiments in our previous paper were performed with saliva collected from only one subject [44]. In this current study, saliva from five individual subjects was pooled prior to carrying out the experiments. The identification of proteins found exclusively using the Sun method and not by the Zhang method could be explained by the fact that in the latter method, the precipitated proteins were resuspended in the coupling buffer. Some proteins may not be readily soluble in this buffer and may have been lost in the subsequent steps. Even for proteins that are solubilized in the coupling buffer, carbohydrates may not be well exposed, and oxidation and the ensuing agarose–hydrazide coupling may be inefficient. The identification of peptides exclusively by the Zhang method (and not by the Sun method) could be because of the hydrophilic nature of glycopeptides and their poor retention on the C18 columns. However, we observed that the number of nonformerly N-glycosylated peptides identified were fewer for the Sun method compared to the Zhang method, i.e., the number of nonglycosylated proteins binding to the resin was reduced (Table 1). For the previous analysis of whole saliva using the Zhang method, we measured 163 nonglycosylated peptides. In the current study using the Sun method, we detected only 14 nonglycosylated peptides in whole saliva. This appears to be a significant advantage of the N-glycopeptide capture method over the glycoprotein capture.

Identification of N-Glycoproteins in Parotid, Submandibular, and Sublingual Fluids

To extend our catalogue of salivary N-glycoproteins, we examined the salivary secretions of the three main salivary glands: PA, SM, and SL. Whole saliva is likely to include contributions from the salivary glands and other sites in the oral cavity. Moreover, the collection and processing of saliva from the PA, SM, and SL glands is slightly different from whole saliva, as the stimulation of secretion by the application of citric acid to the tongue was not used, and the centrifugation of saliva to remove food debris was not required. We applied the Sun method to identify N-glycoproteins from PA, SM, and SL fluids. The total number of formerly N-glycopeptides and the N-linked glycoproteins they represent identified from PA, SM, and SL fluids are 62/34 (peptides/protein), 80/44, and 98/53, respectively (Tables 2, 3, and 4). A comparison of glycoproteins isolated from the three glands is shown in Fig. 3a,b. Only 42 formerly N-glycosylated peptides from 25 N-glycoproteins were found in all three glands. There were a significant number of N-glycoproteins that were unique to the saliva secretions of the individual glands. Figure 4a shows the MS/MS spectrum of a doubly charged formerly N-glycosylated peptide (LNAENN*ATFYFK, m/z 716.8) from splice isoform 1 of kininogen. This formerly N-glycosylated peptide was found only in the PA fluid and not in whole saliva, SM, or SL gland saliva. Figure 4b shows the MS/MS spectrum of a triply charged peptide (HYTN*SSQDVTVPCR, m/z 555.6) from the hypothetical protein DKFZp686C02220. This formerly N-glycosylated peptide was observed in SM saliva but not in whole saliva, PA, or SL gland fluids.

Table 2.

N-linked glycoproteins identified in parotid salivary fluid

IPI accession number	Protein name	MW (Da)	Formerly N-glycosylated peptide sequencea	Formerly N-glycosylated peptide scoreb	Nonformerly glycosylated peptides identified
IPI00004573	Polymeric-immunoglobulin receptor	83,262	AN*LTNFPEN*GTFVVNIAQLSQDDSGR	100	3
			GLSFDVSLEVSQGLGLLN*DTK	74
			IIEGEPNLKVPGN*VTAVLGETLK	118
			LSLLEEPGN*GTFTVILNQLTSR	118
			QIGLYPVLVIDSSGYVNPN*YTGR	101
			VPGN*VTAVLGETLK	115
			VPGN*VTAVLGETLKVPCHFPCK	54
			WN*NTGCQALPSQDEGPSK	48
IPI00011229	Cathepsin D	44,524	GSLSYLN*VTR	68	0
IPI00012887	Cathepsin L	37,540	YSVAN*DTGFVDIPKQEK	42	0
IPI00017601	Ceruloplasmin	122,128	EN*LTAPGSDSAVFFEQGTTR	69	0
			EHEGAIYPDN*TTDFQR	49
IPI00021891	Splice Isoform 1 of Fibrinogen gamma chain	51,479	VDKDLQSLEDILHQVEN*K	53	0
IPI00022417	Leucine-rich alpha-2-glycoprotein	38,944	KLPPGLLAN*FTLLR	78	0
IPI00022431	Alpha-2-HS-glycoprotein	39,300	AALAAFNAQNN*GSNFQLEEISR	113	0
			VCQDCPLLAPLN*DTR	50
IPI00022463	Serotransferrin	77,000	QQQHLFGSN*VTDCSGNFCLFR	100	0
			CGLVPVLAENYN*K	53
IPI00022488	Hemopexin	51,643	ALPQPQN*VTSLLGCTH	73	0
IPI00023673	Galectin-3 binding protein	65,289	ALGFEN*ATQALGR	108	0
			AAIPSALDTN*SSK	58
			GLN*LTEDTYKPR	48
			YKGLN*LTEDTYKPR	35
IPI00025023	Lactoperoxidase	80,237	ASLTN*VTDPSLDLTSLSLEVGCGAPAPVVR	78	0
			IVGYLNEEGVLDQN*R	97
			KPSPCEFIN*TTAR	53
			LRN*LSSPLGLMAVNQEVSDHGLPYLPYDSK	65
IPI00032258	Complement C4-A	192,650	GLN*VTLSSTGR	57	0
IPI00032292	Metalloproteinase inhibitor 1	23,156	FVGTPEVN*QTTLYQR	95	0
IPI00032328	Splice Isoform 1 Of Kininogen	71,900	LNAENN*ATFYFK	71	0
IPI00168728	FLJ00385 Protein (Fragment)	56,111	EEQFN*STFR	51	0
IPI00178926	Immunoglobulin J chain	18,087	EN*ISDPTSPLR	63	0
			IIVPLNNREN*ISDPTSPLR	57
IPI00291262	Clusterin	52,461	LAN*LTQGEDQYYLR	95	0
			MLN*TSSLLEQLNEQFNWVSR	96
IPI00291410	Isoform 1 of long palate, lung, and nasal epithelium carcinoma-associated protein 1	52,408	DHN*ATSILQQLPLLSAMR	33	0
IPI00295105	Carbonic anhydrase VI	35,343	GLN*MTGYETQAGEFPMVNNGHTVQIGLPSTMR	41	0
IPI00296099	Thrombospondin 1	129,330	VVN*STTGPGEHLR	52	0
IPI00296654	Bactericidal/permeability-increasing protein-like 1	49,100	LGATPVAMLHTNN*ATLR	73	0
IPI00298497	Fibrinogen beta chain	55,892	GTAGNALMDGASQLMGEN*R	42	0
IPI00298828	Beta-2-glycoprotein	38,273	VYKPSAGN*NSLYR	79	0
IPI00299729	Transcobalamin I	48,164	ADEGSLKN*ISIYTK	61	0
			MN*DTIFGTMEER	59
IPI00300786	Salivary alpha-amylase	57,731	NVVDGQPFTNWYDN*GSNQVAFGR	112	13
IPI00333140	Delta-notch-like EGF repeat-containing transmembrane	78,438	LVSFEVPQN*TSVK	52	0
IPI00335356	IGHM protein	60,966	GLTFQQN*ASSMCVPDQDTAIR	63	0
IPI00400826	Clusterin	57,796	LAN*LTQGEDQYYLR	96	0
			MLN*TSSLLEQLNEQFNWVSR	96
IPI00418512	DMBT1	78,398	LVNLN*SSYGLCAGR	90	0
			QADN*DTIDYSNFLTAAVSGGIIK	123
IPI00431645	HP protein	31,362	MVSHHN*LTTGATLINEQWLLTTAK	73	0
			NLFLN*HSENATAK	44
			VVLHPN*YSQVDIGLIK	104
IPI00478003	Alpha-2-macroglobulin	163,175	LGHIN*FTISTK	32	0
IPI00550991	Alpha-1-antichymotrypsin	50,596	FN*LTETSEAEIHQSFQHLLR	59	0
IPI00641229	Ig alpha-2 chain C region	53,868	LAGKPTHVN*VSVVMAEVDGTCY	92	1
			LSLHRPALEDLLLGSEAN*LTCTLTGLR	96
IPI00553177	Alpha-1-antitrypsin	46,737	YLGN*ATAIFFLPDEGK	112	0
			ADTHDEILEGLNFN*LTEIPEAQIHEGFQELLR	48
IPI00643034	Splice Isoform 1 of Phospholipid transfer protein	54,705	IYSN*HSALESLALIPLQAPLK	60	0
			VSN*VSCQASVSR	100
			GKEGHFYYN*ISEVK	43
			GAFFPLTERN*WSLPNR	44

^aN* indicates the site of former N-linked glycosylation

^bMascot score listed in parentheses

Table 3.

N-linked glycoproteins identified in submandibular salivary fluid

IPI accession number	Protein name	MW (Da)	Formerly N-glycosylated peptide sequencea	Formerly N-glycosylated peptide scoreb	Nonformerly glycosylated peptides identified
IPI00003351	Extracellular matrix protein 1	60,635	HIPGLIHN*MTAR	62	0
IPI00004573	Polymeric-immunoglobulin receptor	83,262	AN*LTNFPEN*GTFVVNIAQLSQDDSGR	105	2
			IIEGEPNLKVPGN*VTAVLGETLK	108
			LSLLEEPGN*GTFTVILNQLTSR	97
			QIGLYPVLVIDSSGYVNPN*YTGR	127
			VPGN*VTAVLGETLK	116
			VPGN*VTAVLGETLKVPCHFPCK	52
			WN*NTGCQALPSQDEGPSK	45
IPI00010697	Isoform alpha-6X1X2B of integrin alpha-6	126,539	EINSLN*LTESHNSR	31	0
IPI00011229	Cathepsin D	49,768	GSLSYLN*VTR	68	0
IPI00012887	Cathepsin L	37,540	YSVAN*DTGFVDIPKQEK	45	0
IPI00017601	Ceruloplasmin	122,128	EHEGAIYPDN*TTDFQR	63	0
IPI00022431	Alpha-2-HS-glycoprotein	39,300	AALAAFNAQNN*GSNFQLEEISR	41	0
IPI00022488	Hemopexin	51,643	ALPQPQN*VTSLLGCTH	27	0
IPI00023673	Galectin-3 binding protein	65,289	AAIPSALDTN*SSK	58	0
			ALGFEN*ATQALGR	84
			GLN*LTEDTYKPR	60
			EPGSN*VTMSVDAECVPMVR	44
IPI00025023	Lactoperoxidase	80,237	KPALGAAN*R	46	0
			KPSPCEFIN*TTAR	51
			ASLTN*VTDPSLDLTSLSLEVGCGAPAPVVR	96
			IVGYLNEEGVLDQN*R	100
			LRN*LSSPLGLMAVNQEVSDHGLPYLPYDSK	58
IPI00031121	Carboxypeptidase E	53,117	DLQGNPIAN*ATISVEGIDHDVTSAK	49	0
			GN*ETIVNLIHSTR	44
IPI00032292	Metalloproteinase inhibitor 1	23,156	FVGTPEVN*QTTLYQR	96	0
IPI00060143	Protein FAM3D	24,947	GLNIALVN*GTTGAVLGQK	73	0
IPI00166729	Alpha-2-glycoprotein 1, zinc	34,223	FGCEIENN*R	48	3
			DIVEYYN*DSN*GSHVLQGR	66
IPI00168728	FLJ00385 protein (fragment)	56,111	EEQFN*STFR	51	0
IPI00171411	Golgi membrane protein 1	42,917	AVLVNN*ITTGER	70	0
IPI00178926	Immunoglobulin J chain	18,087	EN*ISDPTSPLR	59	0
			IIVPLNNREN*ISDPTSPLR	77
IPI00218413	Biotinidase	61,093	NPVGLIGAEN*ATGETDPSHSK	58	0
IPI00242956	Fc fragment of IgG binding protein	571,718	LLISSLSESPASVSILSQADN*TSK	92	0
			VITVQVAN*FTLR	62
			VTVRPGESVMVN*ISAK	30
			VVTVAALGTN*ISIHKDEIGK	67
IPI00291410	Isoform 1 of long palate, lung, and nasal epithelium carcinoma-associated protein 1	52,408	DHN*ATSILQQLPLLSAMR	31	0
IPI00296099	Thrombospondin-1	129,300	VVN*STTGPGEHLR	70	0
IPI00295105	Carbonic anhydrase VI	35,343	GLN*MTGYETQAGEFPMVNNGHTVQIGLPSTMR	43	0
IPI00296654	Bactericidal/permeability-increasing protein-like 1	49,100	LLAAAN*FTFK	74	0
			LGATPVAMLHTNN*ATLR	68
			SDDNLLN*TSALGR	56
IPI00297910	Tumor-associated calcium signal transducer 2	35,687	HRPTAGAFN*HSDLDAELR	43	0
IPI00299729	Transcobalamin I	48,164	ADEGSLKN*ISIYTK	64	0
			MN*DTIFGFTMEER	92
IPI00298828	Beta-2-glycoprotein 1	38,273	VYKPSAGN*NS		0
IPI00300786	Salivary alpha-amylase	57,731	NVVDGQPFTNWYDN*GSNQVAFGR	79	6
IPI00304557	Short palate, lung, and nasal epithelium carcinoma associated protein 2	26,995	GLNLSFPVTAN*VTVAGPIIGQIINLK	84	2
IPI00328460	Transmembrane protease, serine 11B	46,235	MLNAFQNSSIYK	64	0
			LLPNAN*GSNVQLQLK	80
IPI00328960	Similar to LOC147645 protein	91,568	FVTAGSN*VTLR	69	0
IPI00333140	Delta-notch-like EGF repeat-containing transmembrane	78,438	LVSFEVPQN*TSVK	48	0
IPI00335356	IGHM protein	64,998	GLTFQQN*ASSMCVPDQDTAIR	60	0
IPI00374315	Hypothetical LOC389429	37,902	IILN*QTAR	63	0
IPI00384872	UDP-glucuronosyltransferase 1–7	59,779	YTGTRPSNLAN*NTILVK	46	0
IPI00400826	Clusterin	57,796	LAN*LTQGEDQYYLR	81	0
IPI00418512	DMBT1	193,867	LVNLN*SSYGLCAGR	101
			QADN*DTIDYSNFLTAAVSGGIIK	93
IPI00419215	Alpha-2-macroglobulin like protein 1	161,105	LGHIN*FTISTK	49	0
			QGN*GTFVQTDKPLYTPGQQVYFR	72
IPI00423461	Hypothetical protein DKFZp686C02220	54,126	HYTN*SSQDVTVPCR	55	0
IPI00431645	HP protein	45,177	NLFLN*HSENATAK	39	0
			VVLHPN*YSQVDIGLIK	57
IPI00550991	Alpha-1-antichymotrypsin	50,566	YTGN*ASALFILPDQDK	36	0
IPI00553177	Alpha-1-antitrypsin	46,737	YLGN*ATAIFFLPDEGK	108	0
			ADTHDEILEGLNFN*LTEIPEAQIHEGFQELLR	67
IPI00641229	Ig alpha-2 chain C region	53,868	LAGKPTHVN*VSVVMAEVDGTCY	81	0
			LSLHRPALEDLLLGSEAN*LTCTLTGLR	110
			TPLTAN*ITK	57
IPI00643034	Splice isoform 1 of phospholipid transfer protein	54,705	VSN*VSCQASVSR	90	0
			IYSN*HSALESLALIPLQAPLK	64
IPI00855918	Mucin 5, subtype B, tracheobronchial	590,122	AFGQFFSPGEVIYN*K	80	1
			AQGLVLEASN*GSVLINGQR	141
			DIECQAESFPN*WTLAQVGQK	86
			GN*CTYVLMR	65
			LDGPTEQCPDPLPLPAGN*CTDEEGICHR	24
			LPYSLFHN*NTEGQCGTCTNNQR	20
			QVN*ETWTLEN*CTVAR	68
			VVLLDPKPVAN*VTCVNK	81

^aN* indicates the site of former N-linked glycosylation

^bMascot score listed in parentheses

Table 4.

N-linked glycoproteins identified in sublingual salivary fluid

IPI accession number	Protein name	MW (Da)	Formerly N-glycosylated peptide sequencea	Formerly N-glycosylated peptide scoreb	Nonformerly glycosylated peptides identified
IPI00000877	Hypoxia-upregulated protein	111,266	VFGSQN*LTTVK	79	0
			LSALDNLLN*HSSMFLK	63
IPI00002818	Isoform 1 of kallikrein 11	27,448	CAN*ITIIEHQK	36	0
IPI00003351	Extracellular matrix protein 1	60,635	HIPGLIHN*MTAR	65	0
			HKHIPGLIHN*MTAR	37
IPI00004573	Polymeric-immunoglobulin receptor	83,262	AN*LTNFPEN*GTFVVNIAQLSQDDSGR	106	1
			GLSFDVSLEVSQGPGLLN*DTK	49
			IIEGEPNLKVPGN*VTAVLGETLK	115
			LSLLEEPGN*GTFTVILNQLTSR	114
			QIGLYPVLVIDSSGYVNPN*YTGR	122
			VPGN*VTAVLGETLK	105
			VPGN*VTAVLGETLKVPCHFPCK	82
			WN*NTGCQALPSQDEGPSK	54
IPI00010697	Isoform alpha-6X1X2B of integrin alpha-6	41,000	EIN*SLN*LTESHNSR	32	0
IPI00011229	Cathepsin D	44,524	GSLSYLN*VTR	68	0
IPI00021891	Splice isoform 1 of fibrinogen gamma chain	51,549	DLQSLEDILHQVEN*K	46	0
			VDKDLQSLEDILHQVEN*K	58
IPI00022229	Apolipoprotein B-100	515,241	FVEGSHN*STVSLTTK	29	0
IPI00022431	Alpha-2-HS-glycoprotein	39,300	AALAAFNAQNN*GSNFQLEEISR	104	0
IPI00022463	Serotransferrin	77,000	QQQHLFGSN*VTDCSGNFCLFR	82	0
			CGLVPVLAENYN*K	43
IPI00022488	Hemopexin	51,643	ALPQPQN*VTSLLGCTH	63	0
IPI00022974	Prolactin-inducible protein	16,562	TFYWDFYTN*R	43	0
IPI00023673	Galectin-3 binding protein	65,289	AAIPSALDTN*SSK	58	0
			ALGFEN*ATQALGR	94
			GLN*LTEDTYKPR	39
			YKGLN*LTEDTYKPR	36
IPI00025023	Lactoperoxidase	80,237	ASLTN*VTDPSLDLTSLSLEVGCGAPAPVVR	93	1
			IVGYLNEEGVLDQN*R	110
			KPSPCEFIN*TTAR	74
			LRN*LSSPLGLMAVNQEVSDHGLPYLPYDSK	57
IPI00025846	Splice isoform 1 of desmocollin-2	99,899	NGIYN*ITVLASDQGGR	65	0
			AN*YTILK	32
IPI00027192	Procollagen-lysine	83,497	EQIN*ITLDHR	38	0
IPI00030847	Transmembrane 9 superfamily protein member 3	67,843	IVDVN*LTSEGK	60	0
IPI00031121	Carboxypeptidase E	53,117	GN*ETIVNLIHSTR	44	0
IPI00031547	Desmoglein 3	107,435	LPAVWSITTLN*ATSALLR	80	0
IPI00032258	Complement C4	192,650	GLN*VTLSSTGR	55	0
IPI00032292	Metalloproteinase inhibitor 1	23,156	FVGTPEVN*QTTLYQR	87	0
IPI00060143	Protein FAM3D	24,947	GLNIALVN*GTTGAVLGQK	116	0
IPI00166729	Alpha-2-glycoprotein 1, zinc	34,223	FGCEIENN*R	50	0
IPI00168728	FLJ00385 protein (fragment)	56,111	EEQFN*STFR	51	0
IPI00171411	Golgi membrane protein 1	49,768	AVLVNN*ITTGER	89	0
			LQQDVLQFQKN*QTNLER	43
IPI00178926	Immunoglobulin J chain	18,087	IIVPLNNREN*ISDPTSPLR	62	0
			EN*ISDPTSPLR	55
IPI00242956	IgGFc binding protein	571,718	LLISSLSESPASVSILSQADN*TSKK	40	0
			VTVRPGESVMVN*ISAK	47
IPI00291410	Isoform 1 of long palate, lung, and nasal epithelium carcinoma-associated protein 1	54,878	DHN*ATSILQQLPLLSAMR	46	0
IPI00295105	Carbonic anhydrase VI	35,343	GLN*MTGYETQAGEFPMVNN GHTVQIGLPSTMR	39	0
IPI00296099	Thrombospondin 1	129,330	VVNSTTGPGEHLR	73	0
IPI00296654	Bactericidal/permeability-increasing protein-like 1	49,100	LLAAAN*FTFK	72	0
			LGATPVAMLHTNN*ATLR	66
IPI00297910	Tumor-associated calcium signal transducer 2	35,687	HRPTAGAFN*HSDLDAELR	65	0
IPI00298082	Calcium-activated chloride channel protein 2	101,288	AAN*SSVPPITVNAK	44	0
			HSN*GSYSAFGER	51
			DSFDDALQVN*TTDLSPK	67
IPI00298828	Beta-2-glycoprotein I	38,273	VYKPSAGN*NSLYR	79	0
IPI00299729	Transcobalamin I	48,164	ADEGSLKN*ISIYTK	73	0
			MN*DTIFGFTMEER	96
IPI00300786	Salivary alpha-amylase	57,731	NVVDGQPFTNWYDN*GSNQVAFGR	93	1
IPI00303333	Platelet receptor GI24	33,901	HGLESASDHHGN*FSITMR	41	0
IPI00304557	Short palate, lung, and nasal epithelium carcinoma associated protein 2	26,995	GLN*LSFPVTAN*VTVAGPIIGQIINLK	82	2
IPI00328460	Transmembrane protease, serine 11B	46,235	LLPNAN*GSNVQLQLK	83	0
			MLNAFQN*SSIYK	59
IPI00328960	Predicted: hypothetical protein XP_085831	91,568	FVTAGSN*VTLR	50	0
IPI00332845	Leucine-rich repeats and calponin homology (CH) domain containing 2	69,610	KLPPGLLAN*FTLLR	77	0
IPI00335356	IGHM protein	49,526	GLTFQQN*ASSMCVPDQDTAIR	47	0
IPI00374315	Uncharacterized protein C6orf58	37,902	IILN*QTAR	62	0
IPI00384872	UDP-glucosyltransferase 1–7	59,779	YTGTRPSNLAN*NTILVK	33	0
IPI00400826	Clusterin	57,796	LAN*LTQGEDQYYLR	58	0
			MLN*TSSLLEQLNEQFNWVSR	99
IPI00418512	DMBT1	193,867	LVNLN*SSYGLCAGR	99	0
			QADN*DTIDYSNFLTAAVSGGIIK	92
IPI00419215	Alpha-2-macroglobulin like protein 1	149,965	QGN*GTFVQTDKPLYTPGQQVYFR	72	0
			LGHIN*FTISTK	39
			HSN*GSYSAFGER	51
IPI00431645	HP protein	31,362	VVLHPN*YSQVDIGLIK	53	0
			NLFLN*HSEN*ATAK	41
IPI00550991	Alpha-1-antichymotrypsin	50,596	FN*LTETSEAEIHQSFQHLLR	58	0
IPI00641229	Ig alpha-2 chain C region	53,868	LAGKPTHVN*VSVVMAEVDGTCY	97	3
			LSLHRPALEDLLLGSEAN*LTCTLTGLR	104
IPI00553177	Alpha-1-antitrypsin	46,737	ADTHDEILEGLNFN*LTEIPEAQIHEGFQELLR	51	0
			YLGN*ATAIFFLPDEGK	101
IPI00643034	Splice isoform 1 of phospholipid transfer protein	54,705	GKEGHFYYN*ISEVK	40	0
			GAFFPLTERN*WSLPNR	41
			IYSN*HSALESLALIPLQAPLK	47
			VSN*VSCQASVSR	110
IPI00855918	Mucin 5, subtype B, tracheobronchial	590,122	AFGQFFSPGEVIYN*K	75	0
			AFGQFFSPGEVIYN*KTDR	82
			AQGLVLEASN*GSVLINGQR	137
			DIECQAESFPN*WTLAQVGQK	84
			FGN*LSLYLDN*HYCTASATAAAAR	99
			GN*CTYVLMR	46
			LDGPTEQCPDPLPLPAGN*CTDEEGICHR	28
			LPYSLFHN*NTEGQCGTCTNNQR	31
			QVNETWTLEN*CTVAR	70
			VVLLDPKPVAN*VTCVNK	77

^aN* indicates the site of former N-linked glycosylation

^bMascot score listed in parentheses

Fig. 3.

Comparison of a formerly N-glycosylated peptides and the b N-glycoproteins they represent identified in PA, SM, and SL salivary fluids

Fig. 4.

MS/MS mass spectra of a doubly charged peptide, LNAENN*ATFYFK (m/z 716.8; asterisk denotes the site of N-glycosylation) from splice isoform 1 of kininogen from parotid fluid, and b triply charged peptide, HYTN*SSQDVTVPCR (m/z 555.6) from hypothetical protein DKFZp686C02220 from submandibular fluid

Comparison of N-Glycoproteins in Whole Saliva and Parotid, Submandibular, and Sublingual Fluids

Whole saliva is a complex mixture. It has contributions not only from the three main salivary glands but also from other minor salivary glands located in the mouth. Nonsalivary secretions in whole saliva include gingival crevicular fluid, bronchial and nasal secretions, and blood derivatives that might enter the mouth by cuts or abrasions. Other components of whole saliva include microbes such as bacteria, fungi, and viruses, food, and other extrinsic substances and from the lining of the mouth [51]. It is expected that the glycoprotein profile will differ between that of whole saliva and PA, SM, and SL saliva. A total of 83 formerly N-glycosylated peptides from 46 N-glycoproteins were found to be common in whole saliva and PA, SM, and SL fluids combined. Thirty-nine formerly N-glycosylated peptides from 16 N-glycoproteins were unique to whole saliva, and 34 formerly N-glycosylated peptides from 15 N-glycoproteins were found in PA, SM, or SL and not detected in whole saliva (Fig. 5a,b). Of the glycoproteins that were unique to whole saliva and not found in PA, SM, and SL fluids, three proteins were not detected in our previous global proteome analysis of PA, SM, and SL fluids [52]. These proteins include lumican precursor, complement component C9, and cystatin-related epididymal spermatogenic protein. Of these, complement component C9 has been detected in whole saliva in our previous studies (unpublished data), but the other two proteins were not detected previously in our global whole-saliva proteome analysis. Table 5 is a combined listing of all the salivary N-glycoproteins identified. The detection of N-glycoproteins that are unique to whole saliva is not surprising because whole saliva is a mixture of various components. Some glycoproteins detected in whole saliva may not originate from the salivary glands. Additionally, some proteins originating from the salivary glands may have undergone posttranslational modifications after being secreted into the oral cavity. These formerly glycosylated peptides are observed only in whole saliva. However, the detection of N-glycoproteins unique to PA, SM, and SL saliva is somewhat surprising. It may be explained by the fact that the glycosylation might have been lost upon secretion into the mouth by the action of enzymes. Whole saliva has a plethora of oral bacteria that secrete enzymes to deglycosylate glycoproteins to alter the properties of the protein altogether or to utilize the sugars for their growth [14]. Glycoproteins from the salivary glands may be also sufficiently diluted in whole saliva, so that they fall below the detection limit of our glycoprotein pulldown method. It is also possible that these set of glycoproteins are secreted only upon stimulation of the glands and not otherwise.

Fig. 5.

Comparison of a formerly N-glycosylated peptides and the b N-glycoproteins they represent identified in whole saliva and PA, SM, and SL fluids combined

Table 5.

Combined list of all N-Glycoproteins identified in human salivary fluids

IPI accession number	Protein name	MW (Da)	Formerly N-glycosylated peptidea	Formerly N-glycosylated peptide sequence	Formerly N-glycosylated peptides detectedb
					WS	PA	SM	SL
IPI00000877	Hypoxia-upregulated protein	111,266	VFGSQN*LTTVK	510–520	No	No	No	Yes
			LSALDNLLN*HSSMFLK	822–837	No	No	No	Yes
IPI00002818	Splice isoform 1 of kallikrein 11	27,448	CAN*ITIIEHQK	163–173	Yes	No	No	Yes
IPI00003351	Extracellular matrix protein 1	60,635	HIPGLIHN*MTAR	437–448	Yes	No	Yes	Yes
IPI00004573	Polymeric-immunoglobulin receptor	83,262	VPGN*VTAVLGETLK	466–479	Yes	Yes	Yes	Yes
			WN*NTGCQALPSQDEGPSK	498–515	Yes	Yes	Yes	Yes
			IIEGEPNLKVPGN*VTAVLGETLK	457–479	Yes	Yes	Yes	Yes
			GLSFDVSLEVSQGPGLLN*DTK	118–138	No	Yes	No	Yes
			LSLLEEPGN*GTFTVILNQLTSR	413–434	Yes	Yes	Yes	Yes
			VPGN*VTAVLGETLKVPCHFPCK	466–487	Yes	Yes	Yes	Yes
			QIGLYPVLVIDSSGYVNPN*YTGR	168–190	Yes	Yes	Yes	Yes
			AN*LTNFPEN*GTFVVNIAQLSQDDSGR	82–107	Yes	Yes	Yes	Yes
IPI00007244	Myeloperoxidase	83,815	SCPACPGSN*ITIR	315–327	Yes	No	Yes	No
			SYN*DSVDPR	481–489	Yes	No	No	No
IPI00010697	Isoform alpha 6X1X2B of integrin alpha-6	126,539	EINSLN*LTESHNSR	925–938	No	No	Yes	No
IPI00011229	Cathepsin D	49,768	GSLSYLN*VTR	257–266	Yes	Yes	Yes	Yes
IPI00855918	Mucin 5, subtype B, tracheobronchial	590,122	VVLLDPKPVAN*VTCVNK	4,977–4,993	Yes	No	Yes	Yes
			AQGLVLEASN*GSVLINGQR	136–154	Yes	No	Yes	Yes
			GN*CTYVLMR	5,036–5,044	Yes	No	Yes	Yes
			AFGQFFSPGEVIYN*K	4,888–4,902	Yes	No	Yes	Yes
			AFGQFFSPGEVIYN*KTDR	4,888–4,905	Yes	No	Yes	Yes
			DIECQAESFPN*WTLAQVGQK	1,547–1,567	Yes	No	Yes	Yes
			QVNETWTLEN*CTVAR	4,956–4,970	Yes	No	Yes	Yes
			LDGPTEQCPDPLPLPAGN*CTDEEGICHR	237–264	Yes	No	Yes	Yes
			FGN*LSLYLDNHYCTASATAAAAR	5,054–5,076	No	No	No	Yes
			LPYSLFHN*NTEGQCGTCTNNQR	5,153–5,172	No	No	No	Yes
IPI00012887	Cathepsin L	37,540	YSVAN*DTGFVDIPK	217–230	Yes	Yes	Yes	No
IPI00013972	Carcinoembryonic antigen-related cell adhesion molecule 8		LFIPN*ITTK	284–292	Yes	No	No	No
IPI00017601	Ceruloplasmin	122,128	EN*LTAPGSDSAVFFEQGTTR	396–415	No	Yes	No	No
			EHEGAIYPDN*TTDFQR	129–144	No	Yes	Yes	No
IPI00019943	Afamin	69,024	DIENFN*STQK	28–37	Yes	No	No	No
IPI00020091	Alpha-1-acid glycoprotein 2	23,588	QNQCFYN*SSYLNVQR	87–101	Yes	No	No	No
IPI00020487	Extracellular glycoprotein lacritin	14,237	QFIEN*GSEFAQK	115–126	Yes	No	No	No
IPI00020986	Lumican	38,405	LHINHNN*LTESVGPLPK	121–137	Yes	No	No	No
IPI00021891	Splice isoform 1 of fibrinogen gamma chain	51,479	VDKDLQSLEDILHQVEN*K	62–79	Yes	Yes	No	Yes
			DLQSLEDILHQVEN*K	65–79	Yes	No	No	Yes
IPI00022229	Apolipoprotein B-100	515,241	FVEGSHN*STVSLTTK	3,405–3,419	No	No	No	Yes
IPI00022395	Complement component C9	63,133	AVN*ITSENLIDDVVSLIR	413–430	Yes	No	No	No
IPI00022417	Leucine-rich alpha-2-glycoprotein	38,944	KLPPGLLAN*FTLLR	178–191	Yes	Yes	No	Yes
IPI00022429	Alpha-1-acid glycoprotein 1	2,477	QDQCIYN*TTYLNVQR	87–101	Yes	No	No	No
IPI00022431	Alpha-2-HS-glycoprotein	39,300	AALAAFNAQNN*GSNFQLEEISR	166–187	Yes	Yes	Yes	Yes
			VCQDCPLLAPLN*DTR	145–159	Yes	Yes	No	No
IPI00022463	Serotransferrin	77,000	QQQHLFGSN*VTDCSGNFCLFR	622–642	Yes	Yes	No	Yes
			CGLVPVLAENYN*K	421–433	Yes	Yes	No	Yes
IPI00022488	Hemopexin	51,643	ALPQPQN*VTSLLGCTH	747–762	Yes	Yes	Yes	Yes
			SWPAVGN*CSSALR	181–193	Yes	No	No	No
IPI00643034	Splice isoform 1 of phospholipid transfer protein	54,705	VSN*VSCQASVSR	141–152	No	Yes	Yes	Yes
			GAFFPLTERN*WSLPNR	236–244	No	Yes	No	Yes
			IYSN*HSALESLALIPLQAPLK	395–415	No	Yes	Yes	Yes
			GKEGHFYYN*ISEVK	56–69	No	Yes	No	Yes
IPI00022974	Prolactin-inducible protein	16,562	TFYWDFYTN*R	97–106	Yes	No	No	Yes
IPI00023673	Galectin-3 binding protein	65,289	ALGFEN*ATQALGR	64–76	Yes	Yes	Yes	Yes
			EPGSN*VTMSVDAECVPMVR	188–206	Yes	No	Yes	No
			YKGLN*LTEDTYKPR	394–405	Yes	Yes	No	Yes
			GLN*LTEDTYKPR	396–407	Yes	Yes	Yes	Yes
			AAIPSALDTN*SSK	542–554	Yes	Yes	Yes	Yes
			TVIRPFYLTN*SSGVD	571–585	Yes	No	No	No
IPI00025023	Lactoperoxidase	80,237	ASLTN*VTDPSLDLTSLSLEVGCGAPAPVVR	102–131	No	Yes	Yes	Yes
			IVGYLNEEGVLDQN*R	199–213	Yes	Yes	Yes	Yes
			LRN*LSSPLGLMAVNQEVSDHGLPYLPYDSK	320–349	Yes	Yes	Yes	Yes
			KPSPCEFIN*TTAR	350–362	Yes	Yes	Yes	Yes
IPI00025753	Desmoglein 1	113,644	TGEIN*ITSIVDR	106–117	Yes	No	No	No
IPI00025846	Splice isoform 1 of desmocollin-2	99,899	NGIYN*ITVLASDQGGR	542–557	Yes	No	No	Yes
			AN*YTILK	391–397	Yes	No	No	Yes
			AN*YTILKGNENGNFK	391–405	Yes	No	No	No
			LKAIN*DTAAR	625–634	Yes	No	No	No
IPI00027192	Procollagen-lysine	83,497	EQIN*ITLDHR	194–203	No	No	No	Yes
IPI00027486	Carcinoembryonic antigen-related cell adhesion molecule 5	76,748	TLTLFN*VTR	555–563	Yes	No	No	No
IPI00030847	Transmembrane 9 superfamily protein member 3	67,843	IVDVN*LTSEGK	170–180	No	No	No	Yes
IPI00031019	Cystatin-related epididymal spermatogenic protein	16,265	KLKPVN*ASNANVK	34–46	Yes	No	No	No
IPI00031121	Carboxypeptidase E	53,117	GN*ETIVNLIHSTR	138–150	Yes	No	Yes	Yes
			DLQGNPIAN*ATISVEGIDHDVTSAK	382–406	Yes	No	Yes	No
IPI00031547	Desmoglein 3	107,435	NTGDIN*ITAIVDR	105–117	Yes	No	No	No
			DSTFIVN*K	453–460	Yes	No	No	No
			LPAVWSITTLN*ATSALLR	535–552	Yes	No	No	Yes
IPI00032258	Complement C4	192,650	GLN*VTLSSTGR	1,326–1,336	Yes	Yes	No	Yes
IPI00032292	Metalloproteinase inhibitor 1	23,156	FVGTPEVN*QTTLYQR	46–60	Yes	Yes	Yes	Yes
IPI00032328	Splice Isoform 1 of kininogen	71,900	LNAENN*ATFYFK	289–300	No	Yes	No	No
IPI00060143	Protein FAM3D	24,947	GLNIALVN*GTTGAVLGQK	100–117	Yes	No	Yes	Yes
IPI00166729	Alpha-2-glycoprotein 1, zinc	34,223	DIVEYYNDSN*GSHVLQGR	100–117	Yes	No	Yes	No
			FGCEIENN*R	118–126	Yes	No	Yes	Yes
			FGCEIENN*RSSGAFWK	118–133	Yes	No	No	No
IPI00171411	Golgi membrane protein 1	49,768	AVLVNN*ITTGER	113–124	Yes	No	Yes	Yes
			LQQDVLQFQKN*QTNLER	143–152	Yes	No	No	Yes
IPI00178926	Immunoglobulin J chain	18,087	EN*ISDPTSPLR	70–80	Yes	Yes	Yes	Yes
			IIVPLNNREN*ISDPTSPLR	62–80	Yes	Yes	Yes	Yes
IPI00218413	Biotinidase	61,093	NPVGLIGAEN*ATGETDPSHSK	320–340	No	No	Yes	No
IPI00218460	Splice isoform 3 of attractin	158,537	IDSTGN*VTNELR	411–422	Yes	No	No	No
IPI00242956	Fc fragment of IgG binding protein	571,718	LLISSLSESPASVSILSQADN*TSK	55–78	No	No	Yes	No
			LLISSLSESPASVSILSQADN*TSKK	55–79	No	No	No	Yes
			VTVRPGESVMVN*ISAK	78–95	Yes	No	Yes	Yes
			KVTVRPGESVMVN*ISAK	79–95	Yes	No	No	No
			VVTVAALGTN*ISIHKDEIGK	1,734–1,748	Yes	No	Yes	No
			VITVQVAN*FTLR	2,511–2,522	Yes	No	Yes	No
			YLPVN*SSLLTSDCSER	5,182–5,197	Yes	No	No	No
IPI00400826	Clusterin	57,796	LAN*LTQGEDQYYLR	424–437	Yes	Yes	Yes	Yes
			MLN*TSSLLEQLNEQFNWVSR	352–364	No	Yes	No	Yes
IPI00291410	Isoform 1 of long palate, lung, and nasal epithelium carcinoma-associated protein 1	54,878	DHN*ATSILQQLPLLSAMR	46–65	Yes	Yes	Yes	Yes
			GDQLILNLNN*ISSDR	392–406	Yes	No	No	No
IPI00291488	Splice isoform of WAP four-disulfile core domain protein 2	12,984	TGVCPELQADQN*CTQECVSDSECADNLK	33–60	Yes	No	No	No
IPI00295105	Carbonic anhydrase VI	35,343	LENSLLDHRN*K	247–257	Yes	No	No	No
			GLN*MTGYETQAGEFPMVNNGHTVQIGLPSTMR	65–96	No	Yes	Yes	Yes
IPI00296099	Thrombospondin 1	129,330	VVN*STTGPGEHLR	1,065–1,077	Yes	Yes	Yes	Yes
IPI00296654	Bactericidal/permeability-increasing protein-like 1	49,100	LGATPVAMLHTNN*ATLR	320–336	Yes	Yes	Yes	Yes
			LLAAAN*FTFK	91–100	Yes	No	Yes	Yes
			SDDNLLN*TSALGR	287–299	Yes	No	Yes	No
IPI00297910	Tumor-associated calcium signal transducer 2	35,687	HRPTAGAFN*HSDLDAELR	160–177	Yes	No	Yes	Yes
IPI00298082	Calcium activates chloride channel protein 2	101,288	AAN*SSVPPITVNAK	586–559	No	No	No	Yes
			DSFDDALQVN*TTDLSPK	802–818	Yes	No	No	Yes
			N*VSILIPENWK	75–85	No	No	No	Yes
IPI00298237	Tripeptidyl-peptidase I	61,708	FLSSSPHLPPSSYFN*ASGR	429–447	Yes	No	No	No
IPI00298497	Fibrinogen beta chain	55,892	GTAGNALMDGASQLMGEN*R	377–395	No	Yes	No	No
IPI00298828	Beta-2-glycoprotein I	38,273	VYKPSAGN*NSLYR	155–167	Yes	Yes	Yes	Yes
			LGN*WSAMPSCK	251–261	Yes	No	No	No
IPI00299547	Neutrophil gelatinase-associated lipocalin	22,774	EDKSYN*VTSVLFR	64–76	Yes	No	No	No
			SYN*VTSVLFR	83–92	Yes	No	No	No
IPI00299729	Transcobalamin I	48,164	ADEGSLKN*ISIYTK	209–222	Yes	Yes	Yes	Yes
			MN*DTIFGFTMEER	368–380	Yes	Yes	Yes	Yes
			N*ISIYTK	216–222	No	No	Yes	Yes
			AQKMN*DTIFGFTMEER	365–380	Yes	No	No	No
IPI00300786	Salivary alpha-amylase	57,731	NVVDGQPFTNWYDN*GSNQVAFGR	414–436	Yes	Yes	Yes	Yes
IPI00304557	Short palate, lung, and nasal epithelium carcinoma-associated protein 2	26,995	GLN*LSFPVTAN*VTVAGPIIGQIINLK	122–147	Yes	No	Yes	Yes
			AEPIDDGKGLN*LSFPVTAN*VTVAGPIIGQIINLK	114–147	Yes	No	No	No
IPI00553177	Alpha-1-antitrypsin	46,737	YLGN*ATAIFFLPDEGK	268–283	Yes	Yes	Yes	Yes
			ADTHDEILEGLNFN*LTEIPEAQIHEGFQELLR	94–125	No	Yes	Yes	Yes
IPI00303333	Platelet receptor GI24	33,901	HGLESASDHHGN*FSITMR	117–134	No	No	No	Yes
IPI00328460	Transmembrane protease, serine 11B	46,235	MLNAFQN*SSIYK	81–92	No	No	Yes	Yes
			LLPNAN*GSNVQLQLK	102–116	No	No	Yes	Yes
IPI00328960	Similar to carcinoembryonic antigen-related cell adhesion molecule 1	91,568	FVTAGSN*VTLR	427–437	Yes	No	Yes	Yes
			TPASN*ISTQVSHTK	140–153	Yes	No	No	No
IPI00332845	Leucine-rich repeats and calponin homology (CH) domain containing 2	69,610	MLTYLN*ISR	158–166	No	No	No	Yes
			KLPPGLLAN*FTLLR	178–191	No	No	No	Yes
IPI00333140	Delta-notch-like EGF repeat-containing transmembrane	78,438	LVSFEVPQN*TSVK	215–256	Yes	Yes	Yes	No
IPI00335356	IGHM protein	49,526	GLTFQQN*ASSMCVPDQDTAIR	354–374	Yes	Yes	Yes	Yes
IPI00374315	Hypothetical LOC389429	37,902	IILN*QTAR	66–73	Yes	No	Yes	Yes
			MGMYKIILN*QTAR	61–73	Yes	No	No	No
IPI00384872	UDP-glucuronosyltransferase 1–7	59,779	YTGTRPSNLAN*NTILVK	334–350	No	No	Yes	Yes
IPI00641229	Ig alpha-2 chain C region	55,715	LAGKPTHVN*VSVVMAEVDGTCY	319–340	Yes	Yes	Yes	Yes
			LSLHRPALEDLLLGSEAN*LTCTLTGLR	114–140	Yes	Yes	Yes	Yes
			TPLTAN*ITK	200–208	Yes	No	Yes	No
IPI00418512	DMBT1	259,409	LVNLN*SSYGLCAGR	998–1,111	Yes	Yes	Yes	Yes
			QADN*DTIDYSNFLTAAVSGGIIK	1,298–1,320	Yes	Yes	Yes	Yes
			CSGN*ESYLWSCPHK	822–835	Yes	No	No	No
IPI00419164	Alpha-2-macroglobulin like protein 1	149,965	LGHIN*FTISTK	863–873	Yes	No	Yes	Yes
			QGN*GTFVQTDKPLYTPGQQVYFR	118–140	No	No	Yes	Yes
			HSN*GSYSAFGER	1,018–1,029	No	No	No	Yes
IPI00423461	Hypothetical protein DKFZp686C02220	54,126	HYTN*SSQDVTVPCR	249–262	No	No	Yes	No
IPI00431645	HP protein	31,362	MVSHHN*LTTGATLINEQWLLTTAK	54–77	Yes	Yes	No	No
			NLFLN*HSEN*ATAK	78–90	Yes	Yes	Yes	Yes
			VVLHPN*YSQVDIGLIK	170–186	Yes	Yes	Yes	Yes
IPI00168728	FLJ00385 protein (fragment)	56,111	EEQFN*STFR	285–294	Yes	Yes	Yes	Yes
IPI00550991	Alpha-1-antichymotrypsin	50,596	FN*LTETSEAEIHQSFQHLLR	190–219	No	Yes	No	Yes
			YTGN*ASALFILPDQDK	293–308	No	No	Yes	No

^aN* indicates the site of former N-linked glycosylation

^bWS Whole saliva, PA parotid, SM submandibular, SL sublingual

A gene ontology (GO) analysis of the N-glycoproteins identified in saliva to categorize them according to their cellular location, function, and processes is shown in Fig. 6. The majority of the N-glycoproteins are extracellular (40%; Fig. 6a). Several identified N-glycoproteins were annotated as membrane proteins (14%). Liu and coworkers employed the similar hydrazide chemistry to analyze the human plasma N-glycoproteome and observed a similar trend [42]; the majority of the plasma N-glycoproteins are extracellular or plasma membrane proteins. However, nuclear, cytoplasmic, or cytoskeletal proteins were not identified in the plasma N-glycoprotein study, whereas in our study of salivary proteins, we observed proteins from the nucleus, cytoplasm, and cytoskeleton. Figure 6b shows the GO distribution of salivary glycoproteins based on their functions. Fifty-one percent of the glycoproteins identified are involved in binding, a trend observed also in the analysis of plasma N-glycoproteins [42]. A large number of salivary N-glycoproteins also showed catalytic activity and also a role in enzyme regulation. Salivary N-glycoproteins are involved in catalytic functions such as peptidase, nuclease, hydrolase, and transferase activities. N-Glycoproteins detected in whole saliva are also involved in biological processes such as metabolism, transport, response to stimuli, response to stress, and signal transduction.

Fig. 6.

Gene ontology analysis of the a cellular distribution and b cellular functional distribution of N-glycoproteins identified in salivary fluids

Discussion

The glycoprotein pulldown method using agarose–hydrazide is an efficient technique for identifying N-linked glycoproteins in complex mixtures. We employed this method to study the whole salivary N-glycoproteome by the Zhang method [41, 44] and the Sun method [46]. This aided us to extend our list of whole-salivary N-glycoproteins that may not have been achieved by the application of one method alone. In our study of N-glycoproteins in whole saliva, several proteins were identified by the Zhang method that were not identified by the Sun method and vice versa.

In this current study, we attempted to probe deeper into the salivary proteome and extend the N-glycoprotein list by examining not only whole saliva but PA, SM, and SL fluids. Combining data sets obtained from whole saliva, PA, SM, and SL saliva, we measured 148 sites of N-linked glycosylation to date. Larsen et al. reported 97 sites of N-linked glycosylation in whole saliva [40]. An overlap of 62 sites of N-glycosylation was found in a comparison of the two studies. In a comparison of N-glycoproteins in whole saliva versus PA, SM, and SL saliva, a large number of N-glycoproteins were identified in PA, SM, and SL saliva that were not found in whole saliva and vice versa. In our study, 15 N-glycoproteins were detected in PA, SM, or SM saliva and were not found in whole saliva.

In this study, we report isolation of glycoproteins from the fluids from the SM and SL glands separately. The SM and SL glands are both located below the tongue. It was earlier believed that it is difficult to segregate the two fluids, as they often share a common salivary duct to empty the contents of the gland into the oral cavity [53]. However, other studies have shown that many individuals have separate salivary ducts for the two glands, and it is possible to collect secretions from SM and SL separately [54]. Many new devices have been fabricated that make the collection of segregated SM and SL saliva easier [50, 55]. Hu and coworkers found markers that clearly differentiate SM from SL saliva. They found cystatin C to be a specific marker for SM saliva and Muc5B and calgranulin B for SL saliva [56]. During the course of collection of SM and SL saliva for our experiments, calgranulin B was employed as a marker to ensure purity of the segregated samples. Cystatin C and calgranulin B have no sites of N-linked glycosylation and were thus not detected by our method. However, Muc5B has several known and putative sites of N-glycosylation. In our current study, we found formerly N-glycosylated peptides in SM and SL, but we found a slightly larger number of Muc5B peptides in SL compared to SM; a total of ten formerly N-glycosylated peptides were found in SL and only eight in SM. Hu et al. found no peptides from Muc5B in SM saliva in their shotgun protein identification experiments. However, we did find differences in the overall N-glycoprotein profile between SM and SL saliva (Fig. 3). As a result, we believe that collection of segregated SM and SL fluids can be accomplished and their salivary proteomes can be differentiated.

Studies on the salivary glycoproteins have potential implications on salivary biomarker discovery efforts, as saliva is gaining popularity as a diagnostic fluid for disease markers. Many attempts have been made in the past few years to use saliva to discover biomarkers for diseases localized in the oral cavity or in the head/neck region. Some diseases for which saliva has been used as a medium for biomarker discovery include oral cancer squamous cell carcinoma [57–65], head and neck squamous cell carcinoma [66–68], and Sjogren’s syndrome [69–77]. In addition, saliva has shown promise for use in the detection of many nonoral (i.e., nonproximal) systemic diseases. In patients with breast cancer, elevated levels of C-erb2 and CA15-3 have been detected in saliva of cancer patients versus control subjects [78–80]. Antibodies to the human immunodeficiency virus (HIV) have been found in saliva of HIV-positive patients; saliva-based HIV tests are now gaining popularity [81]. We believe our studies on N-glycoproteins in whole saliva will benefit future work on disease biomarker discovery.