Background/Purpose: Salivary proteomics has recently appeared as a promising tool for the identification of novel diagnostic biomarkers for primary Sjögren’s syndrome (SS). However, to date the vast majority of the studies have focused the attention essentially on a restricted panel of high-abundance proteins, partially limiting the diagnostic potentiality of salivary proteomics. Moreover, the clinical inter-subject variability of SS seem to be reflected in the variability of the proteomic profiling. Aims of this study were: 1) to identify and validate putative salivary biomarkers in SS by using LC-MS/MS. 2) to highlight a specific fingerprint able to discriminate between different SS phenotypes, defined on the basis of the focus score (FS) and on the variation of the salivary flow rate (USFR).

Methods: USFR was collected from 18 patients with SS (AECG criteria, 2002). Six patients presented a high focus score (FS≥3) and normal USFR (group A), six patients presented a FS≥3 and an USFR<1.5 ml/15 (Group B),  and 6 patients a low focus score (FS<3) and USFR <1.5 ml/15 (group C). Six healthy volunteers (CTRL) represented the controls. ProteoPreop Immunoaffinity Albumin and IgG depletion kit (Sigma) was used for high-abundance proteins removal.  A high-throughput liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for the proteomic analysis. Western Blotting (WB) and immunohistochemistry on minor salivary gland biopsies were used to validate proteomics results. Principal component analysis (PCA) was utilized for statistical analysis.

Results: Overall, 100 differentially expressed candidate biomarkers were identified. When compared to CTRL, the salivary proteomic profile of SS patients was characterized by 20 proteins which were significantly decreased and 113 proteins which were significantly increased. Among the differentially expressed proteins, of interest were: proline-rich proteins, cystatins, calcium-binding proteins, antigen binding proteins, profilin and other cell motion-related proteins, proteins involved in apoptosis, defence- and inflammatory-response. PCA distinctively discriminate between CTRL and SS patients stratified as previously described. Preliminary validation by WB and immunohistochemistry of prolactin-inducible protein precursor, cystatins C, S and SA, S-100A7 protein, kallikrein-6 and histhidine-rich glycoprotein indicated similar expression profile trends to those identified by LC-MS/MS. 

Conclusion: The proteomics workflow was able to detect novel candidate biomarkers potentially related to specific phenotypes of SS disease. These candidate biomarkers might be useful to improve SS non-invasive diagnosis and clinical stratification.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/identification-and-validation-of-novel-putative-salivary-proteomic-biomarkers-in-sjogrens-syndrome-and-different-disease-subsets/