Open Access

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

  • Prasanna Ramachandran1,
  • Pinmanee Boontheung1,
  • Eric Pang1,
  • Weihong Yan1,
  • David T. Wong2 and
  • Joseph A. Loo1, 3Email author
Clinical Proteomics20084:9005

https://doi.org/10.1007/s12014-008-9005-0

Received: 27 January 2008

Accepted: 9 May 2008

Published: 17 June 2008

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.

Keywords

Proteomics Mass spectrometry Isoelectric focusing N-linked glycoproteins Whole saliva Parotid saliva Submandibular saliva Sublingual saliva Disease biomarker

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 [46]. 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 [2729]. 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).
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

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

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.
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

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*MTGYETQAGEFPMVNNGHTVQIGLPSTMR

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

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 [5765], head and neck squamous cell carcinoma [6668], and Sjogren’s syndrome [6977]. 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 [7880]. 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.

Declarations

Acknowledgments

We wish to thank Drs. Jimmy Ytterberg, Rachel Ogorzalek Loo, Shen Hu, and Melissa Sondej (UCLA) for their helpful advice. The UCLA Mass Spectrometry and Proteomics Technology Center was established with a grant from the W. M. Keck Foundation. This work was supported by a grant from the National Institutes of Health (National Institute of Dental and Craniofacial Research; U01 DE016275) to DTW and JAL, the Ruth L. Kirschstein National Service Award (GM07185), and the UCLA Fundamental Clinical Research Training Grant (T32 DE007296; to P. R).

Authors’ Affiliations

(1)
Department of Chemistry and Biochemistry, Molecular Biology Institute, University of California—Los Angeles
(2)
School of Dentistry, Dental Research Institute, University of California—Los Angeles
(3)
Department of Biological Chemistry, David Geffen School of Medicine

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