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Saliva an alternative diagnostic biological fluid

There is a growing interest in clinical and translational research for the discovery and development of biomarkers that are indicative of a disease status and progression (1,2). Biomarkers of clinical relevance are predominantly proteins, and the advent of new Mass Spectrometry (MS) technology platforms and the sequencing of the human genome have accelerated protein biomarker discovery, particularly in readily available body fluids such as blood, saliva, and urine (3–5). Human saliva mirrors the body’s health and well-being and most of the biomolecules that are present in blood or urine can also be found in salivary secretions (6). However, biomolecular concentrations in saliva are usually one tenth to one thousandth of the levels found

in blood (7). Sensitive detection technology platforms are therefore required to enable the detection of biomolecules in saliva. Another roadblock to the advancement of salivary diagnostics is the lack of information with regard to the baseline levels (reference ranges) of molecules in saliva within a healthy control group to discriminate the molecular composition and levels during a disease stage. In spite of these impediments, the use of saliva in a clinical setting is on the rise, partly due to our increase in knowledge of the basic biochemistry and physiology of human saliva and also as a result of ease of collection relative to blood for clinical applications (2,8–10). The purpose of this review is to provide an overview of current developments and challenges in the field of saliva proteomic research. We have focussed our review on the potential diagnostic utility

REVIEW ARTICLE

Saliva proteome research: current status and future outlook

Benjamin L. Schulz1, Justin Cooper-White2,3, and Chamindie K. Punyadeera2,3

1School of Chemistry & Molecular Biosciences, St Lucia, The University of Queensland, Brisbane, QLD, Australia, 2Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD, Australia, and 3School of Chemical Engineering, The University of Queensland, Brisbane, QLD, Australia

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

Address for Correspondence: Dr Chamindie Punyadeera, Saliva Translational Research Group, Tissue Engineering and Microfluidic Laboratory, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Old Cooper Road, St. Lucia, 4072, Queensland, Australia. Ph. +61 7 3346 3891, Fax. +61 7 3346 3973. E-mail: c.punyadeera@uq.edu.au

(Received 12 January 2012; revised 12 April 2012; accepted 18 April 2012)

Critical Reviews in Biotechnology, 2013; 33(3): 246–259© 2013 Informa Healthcare USA, Inc.ISSN 0738-8551 print/ISSN 1549-7801 onlineDOI: 10.3109/07388551.2012.687361

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Saliva proteome research

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of salivary proteins and peptides due to the success of proteomic biomarker discovery for blood-based assays. Specifically, we address the importance of controlling biological variation (both direct and indirect) and methodological variation in relation to saliva collection and storage for broad proteomic and clinical applications. In addition, we also provide an overview of post-translation modifications (PTMs) of salivary proteins and proteomic challenges pertaining to protein: protein interactions in saliva.

Human saliva offers several benefits over traditional blood-based biochemical analyses for clinical diagnos-tics: non-invasiveness and stress-free sample collection; easy and multiple sampling opportunities; reduced need for sample pre-processing; minimal risk of contracting infectious organisms such as HPV, HEP-B and HIV and it is an ideal biofluid for third world countries due to the relatively low costs associated with sample collection and processing (11–13). A large number of proteomic studies carried out in the past provide a snap shot of the human saliva proteome (9,14–27). Saliva is a clinically informa-tive biofluid which may be useful for early disease detec-tion, disease prognosis and risk stratification, as well as monitoring treatment response in patients facilitating easy clinical management of diseases. However, most of the current attempts to discern biomolecules in saliva, which are suitable for clinical applications (i.e. high sensitivity and high specificity) are in their infancy, and have not yet been translated from research to the clinic. As an example of an attempt to develop methods to detect ischemic heart disease at an early stage using saliva as a biofluid, researchers have developed rapid immunoassays to mea-sure salivary C-Reactive Proteins levels (CRP (7,28)) (CRP is an acute inflammation marker which is also associated with the development of ischemic heart disease (29–31)) as a non-invasive tool to determine the development of coronary events. Saliva has been used as a biological fluid for the diagnosis and prognosis of oral cancers (32–34), diabetes (12), and autoimmune disorders (35). In addition, researchers have identified biomarkers in saliva for the detection of early-stage pancreatic cancer (36). Streckfus et al. (37,38) measured soluble c-erbB-2Her2/neu levels in saliva collected from breast cancer patients and concluded that c-erbB may have potential use in the initial detection and/or follow-up screening to determine the recurrence of breast cancer. Salivaomics is a publicly available database dedicated to salivary omics-based studies and contains a plethora of information on biology, diagnostic potential, pharmacogenomics and pharmacoproteomics (http://www.hspp.ucla.edu). The clinical utility of human saliva has been recently reviewed (1,2,10,13,34,39). However, large clinical studies are warranted, before these research findings are translated into clinical practice.

Potential challenges to saliva diagnosticsDiagnostic tests based on biological fluids in general utilise blood and/or urine and seldom are esoteric fluids such as saliva, sweat, and tears used. One may even say

that saliva’s popularity has suffered because it lacks “the drama of blood”, the “sincerity of sweat” and the “emo-tional appeal of tears” (40). In terms of obtaining suffi-cient sample volumes for clinical biochemical analysis, sweat and tears pose a major problem and urine lacks the wider acceptance by patients due to personal dis-comfort. Saliva is therefore a favourable alternative bio-logical fluid for biomarker discovery. However, current clinical biochemical use of saliva is scarce. The barriers to widespread implementation of saliva diagnostics are primarily (a) our lack of understanding of saliva physi-ology, most importantly diurnal and circadian variation of molecules in saliva, (b) age, (age-related variations have emerged, with a particular focus on the paediatric age group) gender and genetic differences, (c) lack of understanding of the modes of molecule transportation from blood capillaries into saliva, (d) limited functional characterisation of specific salivary peptides and pro-teins, (e) many proteins present in saliva (i.e. histatins, statherins and proline-rich peptides) are highly poly-morphic and undergo PTMs leading to large inter-indi-vidual and intra-individual variations (41), (f ) the lack of standardization of appropriate saliva sampling collection methods and proper sampling procedures with minimal influence on downstream applications (42,43) and (g) the lack of universally accepted normalisation/reference calibrators. Further adding complexity to the above men-tioned drawbacks is that the composition of saliva can change based on diet and fluid intake (44). It is impor-tant to minimise these variables in a clinical setting by asking participants to refrain from eating or drinking one hour prior to donating a saliva sample to obtain similar baseline values and to report the salivary analyte/protein concentrations as a function of salivary flow rate.

Studies of the correlation between molecular concentrations in blood and saliva have found an excellent concordance comparing salivary and blood levels of ethanol, cortisol, theophylline and antibodies to HIV, but a poor concordance with thyroxine, dihydroepiandrosterone, prolactin, and adrenocorticotrophic hormone (45). It is not always possible to obtain concordance for molecules in saliva and blood simply because of the differences in molecular and biomolecular variations and also leading to proteolytic cleavages resulting in various biochemical forms, PTM modifications and biological half lives. The work from our group has also found a significant correlation between salivary CRP levels to blood CRP, highlighting the diagnostic potential of saliva (28). More importantly, if a collected saliva sample is contaminated with blood due to local inflammation or micro injury to capillaries that are within the oral cavity, this would mean that there is a leakage of blood or plasma into saliva causing falsely high readings (46). Visual inspection of saliva samples is not adequate to detect blood contamination, since micro-injuries to blood vessel membranes may allow red blood cells to pass through at levels too low to be visible but still high enough to cause false positive results. A salivary blood contamination enzyme immunoassay (which measures

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transferrin, a large protein abundantly present in blood and that generally occurs only in trace amounts in saliva) is useful to rule out blood contamination (www.salimetrics.com). These challenges facing saliva must be overcome before saliva will be widely accepted as a diagnostic fluid in a clinical setting.

Saliva production and its regulationHuman saliva is a plasma ultra filtrate and contains pro-teins that are either synthesised in situ in the salivary glands or are derived from blood. The salivary glands are composed of two types of epithelial cells and these are acinar and ductal cells. Saliva is produced in the acinar cells and stored in the salivary granules until an appropriate stimulation occurs. Upon stimulation, the salivary fluid passes from the lumen of the acini cells into a branching network of ducts, where it is collected and enters into the oral cavity. Salivary proteins undergo modifications in the salivary ducts, namely, proteolytic cleavage, partial deglycosylation, and protein-protein complex formation, and hence the saliva that is produced by the glands is different in composition to the saliva that is released into the oral cavity (47). Upon release of saliva into the oral cavity, the fluid is mixed with a number of exocrine, non-exocrine, cellular and exogenous compo-nents, ultimately constituting whole saliva (WS). Human WS represents a mixture of secretions from three major glands (submandibular, sublingual and parotid) as well as minor salivary glands, gingival crevicular fluid (GCF), expectorated bronchial secretions, serum and blood cells from oral wounds, microorganisms, proteins from food debris and desquamated epithelial cells. Therefore, the composition of WS is highly variable depending on the time and the nature of collection (47) and therefore rep-resents a complex balance between local and systemic sources which can be of diagnostic use (48).

Both the volume and the type of saliva secreted are under the control of the autonomic nervous system (49). Quantitative and qualitative changes in salivary secre-tion or composition induced either by systemic or local oral conditions can cause functional deficiencies in saliva (50,51). Studies in animals have demonstrated that secretion of proteins in saliva is under parasympathetic control (increases salivary flow rate) and sympathetic control (decreases salivary flow rate (52–55)). Salivary flow and saliva composition are affected by both internal factors including normal daily changes due to the cholin-ergic and adrenergic systems, and external factors such as medication, radiation and food ingestion (56). Radiation affects salivary gland function leading to a significant reduction in salivary flow rate. This is particularly relevant with head and neck cancer patients who have undergone radiation treatments. These patients have difficulties in swallowing food as a consequence of impaired salivary gland function (57). On average, a person produces 1–2 L of saliva a day (58,59), with an average protein concen-trations varying from 0.5 to 2 mg/mL (60). Salivary flow rate varies throughout the day, with maximum secretion

at around 3–5 pm when the parotid peaks at 1–2 mL/min, and a minimum secretion at around 3–5 am (51,61). Insufficient salivary flow rates and dysfunctional saliva lead to an increase in dental caries, gum disease (gingivi-tis), and or/other oral problems (9).

The functions of saliva within the oral cavity are highly multifaceted and include maintaining homeostasis; promoting wound healing; lubrication of the oral cavity; interacting with teeth surfaces to facilitate their minerali-sation; digestion of carbohydrates by salivary α-amylase (62); digestion of lipid by salivary lipase; and facilitating chewing, speaking, swallowing and taste perception. While the human mouth provides good growth condi-tions for many microorganisms, the anti-microbial prop-erties of saliva aid in clearance and growth inhibition of microorganisms to maintain oral hygiene (37).

Biomolecule transport across the blood capillaries into salivaThe salivary proteome consists of molecules that are synthesized in the salivary glands as well as the proteins that cross the endothelium (blood) and epithelium (salivary gland) cells. There are a number of mechanisms whereby molecules are transported from blood to saliva. Lipophilic molecules including steroid hormones such as testosterone, oestrogens and progesterone are trans-ported into saliva by passive diffusion (63,64), while water and electrolytes filter from blood circulation through the pores of acinar cells. Various peptides from blood are transported through protein channels, while large proteins are transported into saliva via pinocytosis (13). As an example, a molecule such as CRP (115 kDa) is too large to pass from the circulation into the salivary glands by diffusion or ultrafiltration (65), and it is hypothesized to enter into saliva, like many other serum proteins, as a component of GCF (66). For a detailed description of molecular transportation mechanisms refer to our review article (13).

The human salivary proteome

Salivary protein biosynthesis starts with gene transcription and translation of salivary proteins in the glands, followed by post-translational processes that include glycosylation, acetylation, phosphorylation and proteolysis (47,67). A study by Yan et al. (4) compared the human salivary proteome to the plasma proteome using a peptide fractionation method coupled to a cation exchange and MS technique and documented a total of 1939 non-redundant salivary proteins compiled from a total of 19474 unique peptide sequences identified from whole and ductal saliva; 740 of the 1939 proteins were identified in both ductal and whole saliva. The analysis of the plasma proteome revealed a total of 3020 proteins, 597 of which were also found in human saliva. When using a hexapeptide library to compresses the dynamic range of proteins in saliva (to comparatively enrich low abundant proteins), Bandhakavi et al. (68) identified

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2340 salivary proteins using a single analysis platform. About 99% of the total protein content of the plasma proteome is contributed by 22 abundant proteins (14). In contrast, the 20 most abundant proteins in human WS constitute only 40% of the protein content (14). This implies that it should be feasible to detect biomolecules of clinical sensitivity and specificity in saliva with ease as compared to blood. However, the proteome of WS is highly susceptible to a variety of physiological and biochemical processes, unlike the relatively stable plasma proteome, and this presents a challenge for clinical saliva proteomics (47,69–71). The large dynamic range of protein concentrations in saliva is another challenge. For instance, the abundant enzyme α-amylase is present in saliva at mg/mL concentrations, while the IL-6 and IL-8 cytokines of potential clinical relevance are present only at concentrations of pg/mL (72). The saliva proteome also changes as a function of age. A loss of salivary acinar cell function was documented in healthy adults as a consequence of aging (73,74), while salivary production remained age stable in healthy adults. Such effects must be carefully considered in the development of salivary diagnostic assays, primarily by inclusion of appropriate control groups in assay development and validation.

The composition of saliva may be affected by many physiological variables (61), of which the most important factors are the salivary flow rate (75), the type of saliva (e.g. stimulated vs. unstimulated), genetic polymor-phisms (71), nature and the duration of the stimulus, and circadian and circannular rhythms. As an example, sali-vary cortisol levels are highest in the morning, soon after awakening and lowest in the evening and at night and one should take this into consideration when interpret-ing salivary cortisol values (76,77). The secretions from salivary glands have been shown to vary considerably and

the salivary secretions have been reported to be affected by different forms of stimulations (e.g. mechanical or acid stimulation of saliva), including time of day, diet, age, gender, disease status and pharmacoagents (78,79). With respect to the proteinaceous components, saliva is similar to other body fluids, and contains a number of small molecular weight proteins (i.e. below 20 kDa). These small molecular weight proteins are grouped into 6 structurally related major classes: histatins, proline-rich-peptides (acid, basic and neutral), statherins and cys-tatins. The most abundant proteins in saliva are shown in Figure 1A, and the functions of these classes of proteins are summarized in Figure 1B (80). Post-translational modifications of salivary proteins lead to these proteins occurring in structurally related families. As an example, proline-rich peptides fall under acidic and basic families. A better understanding of salivary protein modifications will be a prerequisite to gain insights into the physiologi-cal and pathological processes that reflect a person’s health and well being. Establishing the human salivary proteome has the potential to open up new avenues for disease biomarker discovery and design of targeted therapeutics for co-morbid diseases.

Human saliva consists of a large number of proteins and peptides (the salivary proteome and peptidome (81)), which aids in maintaining oral homeostasis. In addition, both inorganic and organic compound levels in human saliva have been associated with disease diagno-sis and prognosis. For a detailed description of the use of inorganic compounds for disease detection, refer to the review article by Ferguson (61). So far, the association of salivary ion profiles to clinical applications has not proved to be of diagnostic value, except in situations where salivary thiocynate levels have been analysed to rule out smokers from non-smokers (82) and salivary

Figure 1. An overview of the human salivary proteins and their abundance (%). (A) representation of the percentage of proteins present in secreted saliva (B) an overview of the functions of the proteins found in saliva.

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nitrate levels have been analysed to estimate dietary nitrate intakes (83).

Total number of salivary proteins identified by previ-ous researchers is dependent on the methods that they have used (see Table 1 below). These researchers have used a number of protein separation methods to maxi-mize the total number of identified peptides/proteins in saliva. Such separation techniques have been shown to be essential to allow identification of low abundant pro-teins (LAPs) in complex biological mixtures. The ease of identification of proteins in complex mixtures is roughly inversely proportional to the abundance of the proteins. Approaches to allow identification of LAP have largely focused on separation techniques to separate compo-nents of the sample into several fractions, which focuses on separating High Abundant Proteins (HAPs) from LAP to facilitate the detection and characterization of the latter. Recent use of techniques, such as ‘hexapeptide’ libraries to compress the dynamic range (84), has pro-vided another approach enabling the detection of LAPs.

Methodological variations relating to saliva sampling and storage conditionsSaliva proteome biomarker discovery may be influenced by the type of saliva acquired from an individual (whole saliva vs. individual glandular saliva, or unstimulated vs. stimulated saliva (17)). There are commercially available saliva collection devices suited for both life science research as well as for diagnostic purposes, such as DNA Genotek (www.dnagenotek.com); Salimetrics oral swabs (http://www.salimetrics.com); Oasis Diagnostics® VerOFy® I/II; DNA SAL™ (http://www.4saliva.com); OraSure Technologies OraSure HIV specimen collection device (http://www.orasure.com); CoZart® drugs of abuse collection devices (http://www.concateno.com); and the Greiner Bio-One Saliva Collection System (http://www.gbo.com (13)). These

saliva sample collection methods assist in obtaining either unstimulated or stimulated saliva. Saliva collection procedures differ based on the type of saliva that one is interested in collecting. As an example, for ductal secretion collections, one can use Carlson–Crittenden cup (85,86) over the orifice of the Stenson’s duct (87,88). However, these methods are invasive and forfeit the non-invasive advantage of saliva for clinical use. It is important to determine experimentally which collection device is suited for one’s need before commencing any clinical trials. Standardisation of saliva collection methods is also vital in translating saliva research from the laboratory to the clinic.

For the purposes of precision and accuracy in mea-surement, and interpretation of results, it is crucial to know the sources of variability that exert a systematic influence on sampling. Variability can be both biological and methodological in origin, and failure to identify its sources may induce erroneous interpretations of Type I (false positive) and Type II (false negative) errors. Saliva collection, handling and pre-processing steps are vital in promoting saliva as an alternative diagnostic medium to blood (89,90). The advancement and success of pro-teomic studies is attributed to factors such as specimen collection, handling, and processing (such as the com-mon use of protease inhibitors). It is recommended to use plastic low-protein binding tubes to avoid either the adsorption of analytes to the collection vessels and to avoid the release of materials from the vessels that may interfere with downstream analytics. It is important to be consistent when collecting saliva from patients and controls across a group, as well as during each collection visit. This requires standardised saliva collection and processing protocols. For example, it is recommended to discard the initial 2 min of parotid secretions, due to the large inter-individual variability (91). Depending on the molecule of interest (RNA, DNA and/or protein),

Table 1. Total number of human salivary proteins identified using MS approaches. (This list aims to cover a significant portion of the current literature).Total number of proteins identified in human saliva Saliva type Mass spectrometry method

Saliva pre-processing and MS techniques Reference

200 Whole saliva 2D-gel electrophoresis Centrifuged (92)309 Whole saliva Shotgun proteomics and two-

dimensional gel electrophoresis-mass spectrometry (2-DE-MS).

Centrifugation and Pre-fraction using MW cut off

(151)

102 2D-LC-MS Centrifugation and Pre-fraction using MW cut off

(21)

437 Whole saliva Free flow electrophoresis-LC-MS/MS

LC-ESI MS/MS Analysis (152)

1939 Whole saliva and ductal (4)2340 Whole saliva 2D/3D peptide fractionation

couple with capillary LC-MS/MSCentrifuged and dynamic range compression through hexapeptide library

(68)

914, 917 ParotidSubmandula/sublingual

2D/MS centrifugation (153)

1381 Capillary IEF-LC-MS/MS (154)50 Parotid peptides 3D-peptide fractionation (155)

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one should be able to develop standardised methods. As an example, when analysing salivary proteins, it is imperative to keep samples on ice during collec-tion and processing, but when isolating and amplify-ing DNA collected from salivary mouth rinses, it is not critical that the sample processing occurs on ice. A study from Esser et al. (90) has demonstrated that protein degradation in whole saliva is very rapid and this may occur during saliva collection and handling (saliva samples processed at room temperature have increased proteolysis). It is important to minimize artificial changes to saliva post collection if one wishes to analyse the true salivary proteome. One of the ways to minimise saliva sample degradation is to minimise the time that elapses between sample collection, pro-cessing and storage.

Proteolytic activity plays a role in modifying saliva post collection, and this may then result in mislead-ing information about the saliva proteome i.e. incor-rectly identifying a pre-secretory event due to the post secretory proteolytic activity (80). Both endogenous (derived either from the salivary glands or from the exfoliating cells) and exogenous (oral flora) protease activities contribute to the overall proteolytic activity in saliva samples. One strategy to minimise proteolytic activity would be to include a protease-inhibitor cock-tail post saliva collection, however, their use increases the complexity of the sample and brings interferences into the proteomic analysis, especially when the inhib-itors are peptides.

Saliva samples in research projects are often required to be stored for long periods before they can be assayed. For salivary protein analyses, samples are typically collected on ice, centrifuged to remove insoluble material (4,85,92–98) and conserved at tem-peratures below −20°C until analysis. Some authors freeze samples in liquid nitrogen to guarantee a homo-geneous sample freezing and to avoid the problems associated with slow freezing of biologic samples. To highlight the importance of storing saliva samples at −80°C, Messana et al. (80) reported an asymmetric dimethylation of arginine residuals of various salivary peptides when stored for three days at −20°C in con-trast to saliva samples stored at −80°C (80). In addition, long term storage times (22,99) and multiple freezing and thawing cycles (96) should be avoided when han-dling saliva samples, since this may cause some pro-teins to precipitate out of solution. It is also important to note that extended centrifugation of human saliva may be problematic due to the co-precipitation of pro-teins such as cystatins, α-amylase, PRPs and statherin (96). Centrifugation may be avoided in the case of ‘clean’ samples, such as those obtained directly from salivary glands using canniculation. Researchers have also used centrifugation in conjunction with a protein precipitation (e.g. 10% TCA/acetone/20 mM DTT) to prevent the loss of proteins (100,101).

Current sample preparation strategies for salivary proteomicsStandard proteomics can be conceived as either being ‘bottom-up’ or ‘top-down’. Bottom-up strategies aim to improve the detection of proteins in a sample by digesting them to peptides before analysis. Top-down approaches are used for analysis of entire proteins without protease digestion. This analysis provides an unbiased detection of unexpected protein isoforms resulting from sequence polymorphisms, splice variants or PTMs. Bottom-up approaches minimize sample complexity, as the number of modifications on a single peptide will be lower than in an entire protein. This in turn increases sensitivity. Bottom-up approaches can also be used for the identification of PTMs, for instance by releasing glycans from glycoproteins by enzymatic or chemical means prior to analysis. Traditional approaches to proteomics and PTM characterization have used bottom-up approaches, with analysis following protease digestion or PTM release. In saliva, top-down analysis of cystatin SN and PRP3 has identified single nucleotide polymorphisms and identified new sites of phosphorylation (102). Small proteins or peptides are abundant in saliva, and the relatively small size of these components has enabled top-down analytical approaches to profile their abundances and identify PTMs including phosphorylation, Gln to pyro-Glu conversion and glycosylation (103). This study also reported an unusual modification of asparagine with a single N-acetylhexosamine to represent an abundant modification in head and neck cancer patients. This supports the value of top-down non-biased discovery of protein PTMs, and will be useful for analysis of small to medium sized proteins. Both top-down or bottom-up proteomics requires sample preparation to separate or fractionate components before MS detection. These separation procedures can include SDS-PAGE, liquid chromatography, isoelectric focussing, affinity chromatography for depletion or enrichment, and release of PTMs. The order in which these methods are combined can vary depending on the requirements of the sample, downstream analyses, and the experimental question at hand.

In the quest for disease specific biomolecules (which are present at low abundance in human saliva and mainly derived from blood or GCF), it is then imperative to enrich for LAPs by removing HAPs. Enrichment strategies to amplify LAP include pre-fractionation methods, which allow better separation and identification of LAP, such as sequential extraction of proteins with varying buffer condi-tions (104), sub-cellular fractionation (105) and selective removal of HAP via affinity methods (106). Affinity extrac-tion methods include the use of immobilised protein A and anti-immunoglobin support to selectively adsorb immune complexes from patient samples (107); the use of chicken IgY antibodies (108); and the use of combinatorial hexa-peptide ligand libraries (84,109). Combinatorial chemistry-derived hexapeptide libraries CPLLs, ProteomeMiner® (BioRad, USA) have been demonstrated to effectively

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compress dynamic range while increasing proteome cov-erage of complex biological fluids (68,84,110,111).

Post-translational modifications of salivary proteins

The salivary proteome consists almost entirely of secreted proteins that undergo PTMs such as disulphide bond formation, protease cleavage, glycosylation, phos-phorylation, sulfation, and others, are therefore highly abundant (67). Some PTMs are inherent in the process-ing of a particular protein, and as such do not increase structural diversity. Signal peptide cleavage and struc-tural disulfide bond formations are examples of this class of PTMs. In contrast, other PTMs can dramatically increase protein diversity because they may or may not be present (such as in the case of phosphorylation), and/or there may be many different possible structures of the PTM at a given site in a protein (such as in glycosylation). As these modifications are not encoded directly by the genome, it is difficult to predict the precise structure of a PTM, or the extent to which a particular PTM will be present at a particular site on a given protein. PTMs that increase the potential structural diversity of a proteome often regulate protein function and are therefore of par-ticular interest for understanding the status and dynam-ics of biological systems, and in prognostic or diagnostic biomarkers of disease (47). The diversity and regulation of PTMs salivary proteins is underexplored, and we believe this represents a valuable source of potential disease biomarkers.

In order to understand how PTMs allow diversifica-tion and regulation of protein functions, the identity and structure of PTMs on a protein or proteome must first be identified. Such characterisation of a protein’s PTMs is inherently more difficult than identification of the pres-ence of that protein. This is because MS identification of a protein through peptide fragmentation spectral match-ing requires that only one (or a few) peptides from the many potential peptides from a protein be detected with sufficient intensity and quality. However, a priori it is not necessary to know which peptides will be detected. This contrasts with characterisation of the PTMs of a protein. In the case of site-specific PTM identification, a specific peptide must be detected, and identification of a par-ticular peptide provides no information about modifica-tion at other sites. To allow detection of PTMs, a variety of sample preparation and MS detection techniques must be brought to play, with little advance predict-ability of which approaches will prove successful. This makes analysis towards a complete PTM characterisa-tion of a protein challenging, as it requires in depth and non-standard analyses, which is therefore technically challenging, time-consuming and costly. Nonetheless, substantial progress has been made in characterising the many PTMs present on proteins of saliva, and an array of techniques are available for their characterisation. To date, studies have uncovered impressively large PTMs

diversity in a range of salivary proteins, and begun to mine this diversity for biomarkers.

Enrichment strategies for the identification of post-translational modifications in salivaSample preparation methods for low abundant protein detection that focus on sample separation and enrich-ment have proven to be successful and the same is true for PTM detection. Several system level studies aimed at identifying the full suite of PTMs of a particular class have been reported in recent years, with a focus on phosphory-lation (112,113) and glycosylation (114). These successful approaches have relied on the development of efficient enrichment strategies. For instance, in a study to detect N-glycosylation sites on rat proteins, only ~0.5% of all peptides detected from unfractionated samples of whole tissue extracts were glycopeptides. However, enrichment increased this proportion to ~50% (114). Similar results have been reported for other studies of global PTMs.

Enrichment strategies typically use a solid phase matrix with affinity for the PTM of interest. These matrices can be used to enrich either modified proteins or modi-fied peptides. However, since many or even most proteins in saliva are modified, enrichment at the protein level does not substantially increase the proportion of modi-fied proteins, whereas enrichment of modified peptides is efficient. Affinity matrices for enriching glycopeptides and phosphopeptides have been the most successful.

PhosphorylationThe relatively simple chemical nature of phosphorylation allows application of universal enrichment strategies. Phosphopeptide enrichment using IMAC coupled with hexapeptide dynamic range compression and HPLC peptide fractionation has been used in saliva, identifying 217 phosphosites in 85 proteins (98). Other phosphopeptide enrichment strategies include titanium dioxide affinity chromatography (113,115).

GlycosylationGlycan structures are much more structurally diverse than phosphorylation, complicating their enrichment. While no single affinity method can efficiently enrich all classes of glycopeptide, this can also be used to advantage in pro-viding information about the specific glycan structures present on a given peptide. Glycan enrichment matrices can be based on physicochemical or biological affinity.

Physical affinity based on hydrophilic interactions aim to provide maximum scope for enriching diverse glycan structures, but which can consequently suffer from somewhat reduced specificity (116,117). Chemical affinity matrices for glycans include phenyl boronic acid, which forms pH reversible covalent bonds with cis-diols commonly present in glycans (118,119), and hydrazide, which forms nonreversible covalent bonds with glycans pre-oxidized by periodate (120,121). Both of these meth-ods have the advantage of binding glycopeptides through strong covalent bonds, allowing stringent washing to

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increase enrichment specificity. These are also very broad techniques, with essentially all glycans binding to these matrices, irrespective of glycan structure. However, a dis-advantage of hydrazide binding through oxidized glycans is that oxidation destroys the glycan structure, removing the possibility of glycan structural analysis. Glycopeptides must be released by endoglycosidase digest, typically using the rather general PNGaseF enzyme to cleave N-glycans from asparagines, which releases the de-glyco-sylated peptide with a conversion of asparagines to aspar-tate. This conversion can be subsequently used to identify the site of modification. Asparagines modified with gly-cans that are not released by PNGaseF will not be identi-fied by this method. Additionally, glycopeptides modified by O-glycosylation are not amenable to identification with this method, as no enzyme is known which has broad specificity for releasing O-glycans. Glycopeptide enrich-ment by hydrazide coupling after oxidation PNGaseF release has been used to identify 84 N-glycosylation sites from 45 unique proteins in saliva (122). Titanium dioxide has also been used to non-destructively enrich sialylated glycopeptides from saliva, identifying 97 glycosylation sites (123). As sialic acid is a common terminal mono-saccharide on human glycans, this is in general a useful enrichment strategy.

Due to the key functional roles of glycans in biologi-cal systems, many naturally occurring proteins exist with affinity for various glycan structures (124). These lectins have long been used for glycoconjugate detection and enrichment. Glycan binding by lectins is through non-covalent interactions, which are typically weaker than protein-protein interactions. Because of this relatively weak binding, and the proteinaceous nature of lectins, washing of nonspecifically bound peptides cannot be as stringent as with the covalently bound chemical matri-ces. Nonetheless, ~100-fold enrichment of glycopeptides is possible using lectin affinity (114). Lectin-enriched gly-copeptides also still retain their glycans allowing struc-tural analyses. While each individual lectin has a limited and defined glycan-binding specificity, the many differ-ent known lectins have wide ranging glycan specificities. This specificity can also be engineered. Enrichment by selected lectins can be used to provide information on the glycan structures of specific peptides, without the need for more detailed glycan structural analysis (125).

Identification of the site of modification is sufficient for some PTMs, such as phosphorylation, which lack structural diversity. However, for other PTMs such as glycosylation, the structures of glycans on a protein or at a particular site can be of utmost relevance for regulation of protein function, and therefore in biomarker discov-ery. Enrichment of specific glycan structures by lectin affinity can give some information, but while this struc-tural information is readily obtainable, it is inherently unspecific. Standard approaches for glycoproteomic analysis of complex samples generally rely on release of glycans from glycoproteins or protease digestion fol-lowed by analysis of glycopeptides. MS is a key technique

for both of these strategies. Analysis can be performed with varying degrees of pre-separation of components, with glycan release from whole unseparated samples, or following separation by methods including 1D or 2D SDS-PAGE, depending on the detail of characterization desired and sample constraints.

Of particular relevance to glycoproteomic analysis of saliva are mucins, the high molecular weight mucous glycoproteins, which are amongst the most abundant proteins in saliva, and they play key roles in determining saliva’s biological, biophysical and rheological proper-ties. The major mucins present in saliva are MUC5B, MUC7, and the mucin-like protein salivary agglutinin (126). Glycomic or glycoprotomic characterisation of mucins is hampered by their extremely large size (up to ~5 MDa), which makes their separation by tradi-tional SDS-PAGE very difficult. Separation by agarose or agarose-polyacrylamide composite gels (SDS-AgPAGE) has been used successfully to resolve these components prior to glycan release (127–128).

N-glycans can be released from asparagines using PNGaseF, which is a general endoglycosidase, cleaving almost all N-glycan structures (120). PNGaseF released glycans are reducing, and can be analyzed directly (129) or labeled for increased sensitivity. As no enzyme exists which cleaves all O-glycan structures, chemical release strategies such as reductive alkaline β-elimination (127) are commonly used. This method releases all structures of O-glycans, but as it requires harsh reaction conditions it has the potential to modify glycan structures and reducing-end labeling is not possible. Alternative strategies to allow labeling by not reducing the terminal GalNAc monosaccharide have been reported, but are more technically difficult with somewhat lower reproducibility (130). Glycans can be analyzed directly or derivatised, for instance with permethylation, for increased sensitivity and ease of structural characterization (131).

Several MS strategies are commonly used for analy-sis of N- and O-glycans, including positive and negative mode MALDI, C18 LC-ESI in positive mode, or graphi-tized carbon LC-ESI in negative mode (132–134). These analyses have been successfully performed on a variety of MS instruments. Analysis of glycan MS/MS data are not as automated as for peptide MS/MS data. This is because of the comparatively huge diversity inherent in possible glycan structures increasing the required search space, and the non-template driven biosynthesis of glycans not allowing direct comparison with a set sequence database. Nonetheless, several software approaches for glycan struc-tural characterization, and databases of glycan structures with associated MS/MS spectra, are available (135–139).

Glycan structural determination of glycans released from separated proteins is a very useful step, but detailed description of glycoproteins requires that their site-specific glycosylation also be identified. To this end, glycopeptide analysis is becoming a common approach (117,140,141). This strategy can analyse peptides and glycopeptides in a single LC-MS analysis, or after

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enrichment of glycopeptides (see above). As detection of a specific glycosylation site is often difficult to pre-dict in advance, targeted detection of a particular site can require use of several different proteases and MS detection technologies.

Glycosylation analysis of saliva has tradition-ally focused on the abundant mucin and mucin-like glycoproteins, and analysis of these has resulted in results of interest for biomarker discovery. Truncated Tn and sialyl-Tn O-glycan antigens have been associ-ated with salivary gland carcinomas (142). Analysis of salivary mucin glycan structures has also shown high population diversity due to blood group and secretor status (143), emphasizing that caution is required in experimental design when studying these molecules as potential biomarkers. However, studies of mucin glyco-sylation in sputum have reported consistent alterations in structures with prognostic potential (144,145). There seems to be strong potential for identification of glycan prognostic or diagnostic biomarkers from saliva.

SulfationSulfation of tyrosines has been reported on salivary statherin and histatin 1 (146,147). As tyrosylprotein sulfotransferase is present in human saliva (148), it is possible that there are other as yet unidentified sub-strates, and that this modification has a regulatory function and therefore biomarker potential.

Implications of protein: protein interactions for saliva proteomicsMoving from identification of proteins and charac-terization of PTMs to quantification of component abundance, proteomic studies are also moving to identify physical protein-protein interactions. Such interactions are present along a spectrum of strengths, from weak and transient to covalent, and in saliva can be mediated by specific protein-protein interac-tions, glycan recognition and nonspecific aggrega-tion in micelles (47). Some interactions occur during protein synthesis before secretion from the glands, including MUC5B oligomerisation through intermo-lecular disulfide bonds. Other interactions occur only in whole saliva, such as micelle aggregate formation, attachment of MUC7 to bacteria and/or lymphocytes and attachment of proteins and protein aggregates to the tooth pellicle. As centrifugation is a common early step in sample preparation for proteomics of saliva (see Methodological variations relating to saliva sam-pling and storage conditions Section), many of these aggregates are removed from the pool of proteins to be analysed. However, the specific inter-molecular inter-actions that form the basis of these aggregates may contain useful biological information. Such interac-tions remain understudied in saliva, and due to their probable biological significance represent a pool of biomarker candidates.

Selected-reaction-monitoring in MS

After detection of proteins in a sample, and characterization of their PTMs, a proteomics workflow must move towards quantitative or semi-quantitative comparison of samples, either for biological understanding or clinical utility. Selected-Reaction-Monitoring (SRM) MS has shown in recent years to be the premier tool for these analyses. SRM-MS (reviewed in detail elsewhere (149,150)) uses triple quadrupole MS instruments to provide exquisite specificity and sensitivity of detection of pre-defined analytes. SRM-MS has a long history of use in small molecule analysis, and has recently and rapidly become the method of choice for quantitative proteomic studies. The specificity of SRM experiments is obtained through using the three consecutive quadrupoles of a MS instrument to target a specific peptide parent ion in the first quadrupole, fragment that ion in the second quadrupole, and detect a specific fragment ion in the third quadrupole. The combination of parent and fragment ions is typically unique in a given sample, although care must be taken with experimental design to ensure this is the case. Modern MS instruments also typically allow scheduled SRM, which provides additional specificity by targeting a single ‘transition’ for only a given period of an LC separation. Scheduled SRM also allows for more transitions to be performed in a given experiment. However, SRM-MS requires pre-knowledge of the components of a sample. Current MS technology for SRM, while impressively allowing quantitative information on ~6000 components in a 60 min LC run, is not sufficient to characterise the various PTM isoforms present in a fluid such as saliva. In addition, the requirement to predefine targets for quantification precludes the ability to identify unexpected components. This is of key importance for clinical biomarker discovery, where the presence in saliva of a protein not normally present, or an unexpected PTM, is quite possibly the most specific biomarker for a particular condition.

Complete characterisation of protein PTM isoforms, including quantitative information on their occupancy, and protein isoform concentration, is technically very challenging. In fact, complete characterisation is defined by the technological limitations of the day. The extent of characterisation is also proportional to the analysis time required. The most successful uses of saliva for clinical biomarker detection have been for diseases physically associated with the mouth, or when the biomarker was previously known from other sources. Despite continual improvements in sample preparation, MS technology and data analysis, this is likely to remain the case.

Saliva proteomics: future outlook

Current research has used human saliva for disease diagnosis, prognosis as well as risk stratifications. Based on our review, we believe that recommendations can be made with regard to saliva sampling to ensure a practical

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protocol and increase the quality of future studies in the emerging field of saliva clinical proteomics. Prior to saliva being used as a diagnostic fluid, it is necessary to under-stand the (i) normal biological variation of biomolecules in saliva (e.g. diurnal variation, within-subject variation and between-subject variations, age and gender effects) (ii) inter-individual biological variation of biomolecules in saliva (e.g. lifestyle choices, diet, medication, smoking, alcohol and physical activity status and (iii) method-ological variations caused by saliva sampling, handling and storage conditions (iv) methodological variations due to the analytical techniques used. Since the salivary proteome is sensitive to both extrinsic and intrinsic fac-tors, care should be exerted when documenting the con-centration of analytes in saliva. As an example, reference intervals for salivary proteins should take into account diurnal variation of analytes, age, gender etc. In addi-tion it is also important to standardize saliva collection methods to fulfill the requirements of routine analysis translating to clinical settings. Hence, we suggest that methods used in a research environment align as closely as possible with the requirements of clinical application.

Saliva proteomic studies have catalogued a remarkable diversity of proteins and modifications. However, clinical proteomics of saliva is still at a primitive stage, especially with respect to clinical translation. Barriers to the transla-tion of salivary research findings to the clinic include the lack of large clinical studies validating the sensitivity and specificity of disease specific biomolecules; lack of stan-dardized saliva collection and processing protocols; the dynamic and highly variable nature of the saliva proteome; and the lack of protein detection technology platforms to detect the extremely low concentrations of some biomol-ecules of clinical relevance in saliva. Further development in saliva sample preparation and enrichment strategies and MS instrumentation will certainly enable the detection of even lower abundant species in the future. However, the promise of proteomics lies in providing an accurate description of the state of the proteome, and being able to use that information to further understand the biological systems, and for its use in clinical applications. Towards these goals, identification of the presence of proteins is not sufficient. Rather, these aims require both a detailed characterization of the many post-translational protein modifications that add structural and functional diversity to proteome, and quantitative comparisons of the abun-dances of proteins and their modified isoforms between biological or clinical samples. Increased understanding of the components of the saliva proteome, their modifica-tions and dynamics in health and disease will allow saliva to enter the clinical laboratory as an alternative biological fluid for disease prognosis and diagnosis.

Declaration of interest

The authors would like to acknowledge the financial support of the Queensland Government Smart Futures Fellowship Programme (QGSFF), the University of

Queensland New Staff Research Funds (UQNSRSF 601252) and the University of Queensland Foundation Research Excellence Award Scheme.

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