Proteomic analysis of saliva: a unique tool to distinguish primary Sjögren's syndrome from secondary Sjögren's syndrome and other sicca syndromes

Abstract

IntroductionA growing interest has arisen in salivary proteomics as a tool for the identification of biomarkers for primary Sjögren's syndrome (pSS). Nonetheless, only a limited number of preclinical validation studies have been performed, limiting the possibility of translating proteomic results into clinical practice. The primary aim of this study was to refine the diagnostic power of a panel of candidate salivary biomarkers described in pSS with respect to both healthy volunteers and pathological controls. We also explored the pathogenetic function of the detected putative biomarkers both in the local exocrinopathy and in the systemic inflammatory processes of SS.MethodsOne hundred and eighty patients were included in the study overall. In the first "exploratory phase", we enrolled 40 females with pSS, 40 sex- and age-matched healthy volunteers, 10 patients with sicca non-SS and 15 secondary SS (sSS) patients. The testing cohort of the second "challenge phase" of the study was represented by 75 unselected, consecutive subjects: 19 pSS, 21 healthy volunteers, 10 sicca non-SS and 25 sSS patients. Salivary proteomic analysis was performed combining two-dimensional electrophoresis (2DE) and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). Western blot (WB) analysis and enzyme-linked immunosorbent assay (ELISA) were employed to validate 2DE results. Ingenuity Pathway Analysis (IPA) Knowledge base was adopted to associate candidate biomarkers in a signalling pathogenetic network.ResultsA total of 28, 6, 7 and 12 protein spots were found to be significantly different in pSS samples with respect to healthy volunteers, non-SS sicca syndrome, SSc-sSS and rheumatoid arthritis-sSS, leading to the identification of 15 differently expressed proteins. Among them, α-amylases precursor, carbonic anhydrase VI, β-2 microglobulin, glyceraldehydes-3-phosphate dehydrogenase (G3PDH), epidermal fatty acid binding protein (E-FABP) and immunoglobulin k light chain (IGK-light chain) apparently showed the most significant differences in pSS when compared to healthy volunteers and non-SS pathological controls. On the other hand, as expected, pSS and sSS salivary profiles shared a great number of similarities.ConclusionsThis study demonstrated that salivary fluid might represent a novel ideal milieu for the detection of a diagnostic panel of candidate biomarkers for pSS, and to gain an insight into the pathogenetic processes underlying glandular and systemic autoimmune disorders.

Beta-2 Microglobulin in Whole Unstimulated Saliva Can Effectively Distinguish Between Sjögren’s Syndrome and Non-Autoimmune Sicca Symptoms

“…This is in contrast to the results from Baldini et al 19 but similar to that reported by Asashima et al 5 Unlike other investigators, 5,18 we only included patients with sicca symptoms, since the idea behind our study was to develop an additional tool to benefit in the diagnosis of those in whom SS is suspected (i.e., in those with sicca symptoms). In our study, fibromyalgia was more common in those with sicca symptoms but no autoimmune disease (snSS group).…”

“…Our snSS group represents such patients. Baldini et al 19 also included a snSS group in their study. Specifically addressing this point, the ROC curve analysis showed an area under the curve of 0.648 (95% CI: 0.559-0.730, p=0.0023) with an optimal cutoff value of 0.638 ng/mL (not shown in Figure 1).…”

Automated Mass Spectrometry–Based Functional Assay for the Routine Analysis of the Secretome

“…Although the principle is simple, this method is not very successful, simply because many eukaryotic cells secrete proteins that are intensely posttranslationally modified. [40][41][42][43][44][45][46][47] Therefore, a peak in the MALDI-MS spectrum does not represent a protein or a peptide but rather a posttranslationally modified protein (or peptide). [45][46][47] However, even if the differences detected are not at the "naked" protein level, the differences observed may still be detected and the proteins responsible for them can be identified.…”

Analysis of the extreme diversity of salivary alpha-amylase isoforms generated by physiological proteolysis using liquid chromatography–tandem mass spectrometry

“…Catalytically active salivary amylase has been reported to bind to microorganisms and tooth enamel, and as such is perhaps crucial in mediating adherence of pathogens to oral surfaces, and allowing microbial growth through provision of glucose from the breakdown of dietary glucose polymers [7][8][9][10]. Changes in the levels of salivary amylase have been reported in many proteomic studies, including implications in periodontitis [3], Sjögrens Syndrome [5,11], sensory perception of bitter tastes [12], and in binding of salivary tannins [13][14][15][16]. It has also been reported that α-amylase is subject to a very high level of posttranslational modification.…”

“…Regulation of this variability is crucial for homeostasis of the oral cavity and hence in oral health. Alterations in the protein composition of saliva have also been associated with diverse diseases, including periodontitis [3], oral cancers [4], and autoimmune diseases such as Sjögrens Syndrome [5]. The changes in salivary protein composition observed in these states may contribute to the progression of these conditions, and may also be of use for identification of biomarkers for disease prognosis or diagnosis [2].…”