Blood has historically been used as the primary routine diagnostic medium for analyzing cellular, molecular and chemical changes associated with pathological conditions. However, the exploration of saliva as a diagnostic fluid has recently gained momentum due to its easy accessibility and non-invasive collection procedures that require little equipment and training. Notably, systemic diseases may cause changes in salivary constituents; e.g. increased levels of proteins such as cytokines and interleukins in Sjögrens syndrome (SS) 33
. One important consideration not always adequately controlled for in past studies is that many diseases are age dependent 34–36
. Consequently, the present study was designed to identify the differentially and commonly expressed salivary proteins in healthy young and older females as an initial step towards the development of saliva-based clinical tests.
This is the first comprehensive, high-throughput proteomic study to identify and quantify the global changes in the abundance of salivary proteins associated with normal aging in healthy females. Tandem liquid chromatography-mass spectrometry methods have a significant advantage for resolving proteins and peptides over 2-dimensional gel electrophoresis methods 37–39
. In the present study HCIC was used for the separation of proteins (see Materials and Methods). Consequently medium and low copy proteins were unmasked from abundant proteins like amylases and histatins, which were identified using an LC-ESI-MS/MS approach that significantly enhanced its identification using Multidimensional Protein Identification technology (MudPIT) 23
. The relative abundance of the identified salivary proteins was quantitated by G-test spectral counting. To increase confidence of our protein identifications, protein identities were deemed acceptable only when two unique peptides were detected for each protein. The overall false positive rate at the protein level was below 5%.
More than 900 proteins were recently identified in parotid saliva by mass spectrometry 18
, 349 of which overlapped with the 532 proteins detected in the present study. Although many proteins were common to both age groups, a surprisingly large number of proteins (50%) were uniquely expressed in an individual age group. The highest number of proteins was identified in the older age group (). It is interesting to note that the majority of the 183 proteins that did not overlap with the previously catalogued parotid saliva proteome 18
, were identified in the older age group, study subjects that were under represented in this earlier study. Consequently, our results significantly expand the number proteins in the human parotid saliva proteome and emphasize that it is important to consider normal, age-related changes in protein abundance when using saliva as a diagnostic fluid.
One of the primary functions of saliva is to protect the upper gastrointestinal tract from infection. Accordingly, the largest number of proteins identified in both age groups was those associated with immunological protection (~45%; Defense and Immune component categories). These immune-related proteins could be further subdivided into proinflammatory, anti-inflammatory and antimicrobial proteins. Common to the saliva in two age groups, the immunoglobulin-related proteins, members of the immunoglobulin superfamily (IgSF) such as Ig kappa chain V-III region VH and kappa light chain, have a role in immune protection 40
. Salivary IgA was the predominant external secretory immunoglobulin isotype. Secretory IgA acts as the first line of defense against pathogens which colonize or invade mucosal surfaces 41
. The high valency of SIgA enables it to agglutinate bacteria, neutralize viruses, enzymes and toxins 42, 43
. SIgA antibodies generated in response to oral administration of bacteria have been detected in the saliva of animals and humans 44, 45
, suggesting their importance as a key factor in maintaining the balance of the oral microflora 46
. IgA1 was previously reported to be more abundant in older individuals than younger 47
, consistent with our results (Table 3; spectral counts, group 1 = 1870; group 2 = 2320). Changes in the abundance patterns of immune related proteins highlight the complexity of the immunoprotection system. Such changes in abundance are hypothesized to occur in response to alterations in the oral microbiota with age, leading to a change in the host’s immune response to a microbial challenge 48
. Indeed, the increase in the total number of proteins in group 2 was partially due to the identification of nearly 2-fold more immune related proteins than in group 1 ().
A significant number of salivary proteins with a primary role in host defense were also identified in the saliva of both the age groups. For example, antimicrobial proteins (AMPs) such as lysozyme, lactoferrin proline-rich proteins, mucins and histatins, which protect the oral cavity by limiting growth or killing bacteria directly, were common to both age groups. The abundance of antimicrobial proteins such as proline rich proteins, mucin-7 and bacterial permeability membrane proteins did not change with age, while lysozyme, lactoferrin, histatin-1, serotransferrin, mucin-5 and NGAL abundance increased in the older age group. In contrast, other antimicrobial proteins such as granulins and jacalin-like lectin decreased with age (group 1>2). In total, 33 proteins from groups 1, and 34 proteins from group 2 were identified as antimicrobial proteins. Using a pair-wise comparison between the groups, 19 (1 vs. 2) host defense proteins were differentially expressed. Of the host defense proteins, only 9 proteins were expressed in both age groups.
Changes in ovarian hormone levels during puberty, pregnancy, menstrual cycle and oral contraceptive use appear to correlate with decreases in the abundance of some antimicrobial proteins responsible for regulating the oral microflora, thereby compromising their protective role and allowing some bacteria to thrive 49
. However, the older subjects (group 2) in this study are all post-menopausal and not receiving hormone replacement therapy. Consequently, changes in AMPs abundance between the young and old groups could be in response to changes in microflora that have been observed with age 50, 51
and/or hormonal status 49
. Regardless of the mechanism, our results demonstrate that the abundance of AMPs changed with age; such changes may play an important role in maintaining homeostasis of the oral microflora.
We found that there was a modest, but significant, decrease with age () in Carbonic anhydrase VI (CA VI) which is responsible for maintaining pH homeostasis in the oral cavity and upper alimentary tract. This enzyme is stored in the secretory granules of the acinar cells of the parotid gland, and its secretion in saliva follows a circadian cycle 52
. CA VI plays a significant role in preventing caries 53
and upper GI ulcers 54
by catalyzing the conversion of salivary bicarbonate and hydrogen ions released by the microbe to carbon dioxide and water 55
. Consequently, one potential mechanism for the increased development of caries lesions in the age may be due to a decrease in the expression of CA VI in this population 56
The abundance of pro-inflammatory molecules such as S100 family, calgranulins A and B also changed with age (Supplementary Table 3
). These proteins are commonly implicated in acute-phase and chronic inflammatory responses 57
. In contrast to the S100 family and the calgranulins, no age-related changes in the abundance of anti-inflammatory molecules such as interleukin 1 and heat shock protein were observed, even though these proteins play a vital role in the down regulation of an immune response. Thus, the detection of a wide range of pro- and anti-inflammatory response molecules, along with antimicrobial proteins, in saliva suggests their importance and correlates with the changing immunological needs of the oral cavity.
Alpha-enolase abundance was elevated in group 2 subjects relative to group 1. Alpha-enolase is a multifunctional glycolytic enzyme involved in a number of activities, such as growth control, hypoxia tolerance, and allergic responses. Notably, α-enolase was also identified as an autoantigen in Hashimoto encephalopathy 58
and lymphoid hypophysitis 59
, and its expression was dramatically increased in the saliva of Sjögren’s syndrome subjects 60
. Consequently, autoantibodies produced by B cell in response to this autoantigen can potentially initiate tissue injury as a result of immune complex deposition 61, 62
A range of cysteine protease inhibitors were also identified in both age groups. Cysteine protease inhibitors are present in saliva, tears and plasma 63
and play a key role in inflammatory and immune responses 62, 63
. Cystatin D, cystatin SA and cystatin B were more abundant in group 2, while cystatin SN and cystatin C, proteins thought to regulate protein degradation at the site of inflammation or to prevent microbial infection 66
, were more abundant in group 1. Two other enzymes involved in the maintenance of healthy tissue, cathepsin B and α 1 antitrypsin, were detected. Changes in their expression have been linked to malignant changes in breast tumor in females 67
. In addition, secretion of the enzyme mediator 14-3-3 protein was significantly more abundant in group 2. 14-3-3 is involved in actin disassembly following an increase in the level of cytosolic Ca2+
and activation of protein kinase C 68
. It also takes part in the regulation of apoptosis and the p53 signalling pathway, where p53 protects cells from stress-induced apoptosis.
In summary, this is the first high-throughput proteomic analysis of parotid saliva to document the proteins expressed in a healthy cohort of young and older adult females. The approach used in our study led to the confident identification of more than 530 proteins, including 183 proteins not previously detected in the parotid proteome 18
. Extensive age-associated changes in the abundance of many of these proteins were noted, especially for proteins associated with host defense mechanisms. Such changes in host defense proteins may reflect the dynamic nature of the very complex milieu of proteins found in saliva and their role in protection against oral microorganisms. In the present aging study, we controlled for gender, age, time of collection and state of menstruation (pre- and post-menopausal, groups 1 and 2, respectively). However, it is difficult to discriminate between bona fide age-related changes in salivary proteins from those induced by numerous other factors such as dietary, alcohol habits, periodontal disease, hydration and hormonal status. Consequently, this study provides an initial database of proteins to evaluate in future studies the effects of additional important factors on saliva composition, and to compare saliva from healthy and disease subjects.