Proteomics Analysis of Tears and Saliva From Sjogren’s Syndrome Patients

Abstract

Sjogren’s syndrome (SS) is characterized by dysfunctional mucous membranes and dysregulated moisture-secreting glands resulting in various symptoms, including dry mouth and dry eyes. Here, we wanted to profile and compare the tear and saliva proteomes of SS patients to healthy controls. Tear and saliva samples were collected and subjected to an isotopic dimethylation labeling shotgun proteomics workflow to identify alterations in protein levels. In tear samples, we identified 83 upregulated and 112 downregulated proteins. Pathway enrichment analysis of the changing proteins by Metascape identified leukocyte transendothelial migration, neutrophil degranulation, and post-translation protein phosphorylation in tears of SS patients. In healthy controls’ tears, an enrichment for proteins related to glycolysis, amino acid metabolism and apoptotic signaling pathway were identified. In saliva, we identified 108 upregulated and 45 downregulated proteins. Altered pathways in SS patients’ saliva included cornification, sensory perception to taste and neutrophil degranulation. In healthy controls’ saliva, an enrichment for proteins related to JAK-STAT signaling after interleukin-12 stimulation, phagocytosis and glycolysis in senescence were identified. Dysregulated protease activity is implicated in the initiation of inflammation and immune cell recruitment in SS. We identified 20 proteases and protease inhibitors in tears and 18 in saliva which are differentially expressed between SS patients and healthy controls. Next, we quantified endogenous proteoglycan 4 (PRG4), a mucin-like glycoprotein, in tear wash and saliva samples via a bead-based immune assay. We identified decreased levels of PRG4 in SS patients’ tear wash compared to normal samples. Conversely, in saliva, we found elevated levels of PRG4 concentration and visualized PRG4 expression in human parotid gland via immunohistological staining. These findings will improve our mechanistic understanding of the disease and changes in SS patients’ protein expression will help identify new potential drug targets. PRG4 is among the promising targets, which we identified here, in saliva, for the first time.

Multi-Omic Biomarkers for Patient Stratification in Sjogren’s Syndrome—A Review of the Literature

“…Tear proteomic analysis by Das et al [ 20 ] using high performance liquid chromatography (HPLC) and mass spectrometry revealed the upregulation of 83 proteins and downregulation of 112 proteins in pSS patients compared to HCs. Enrichment pathway analysis of upregulated proteins included leukocyte trans-endothelial migration, protein-lipid complex remodelling and collagen catabolic pathway.…”

“…However, none of the protein biomarkers correlated with saliva flow rates in pSS. A more recent study by Das et al [ 20 ] identified the upregulation of 104 proteins and downregulation of 42 proteins in pSS compared to controls. Some enriched pathways in patients’ saliva included JAK-STAT signalling after IL-12 stimulation, superoxide metabolic process and phagocytosis.…”

Endogenous production of hyaluronan, PRG4, and cytokines is sensitive to cyclic loading in synoviocytes

“…Conditioned media samples from 24 hours after the completion of loading were analyzed for PRG4 concentration using the AlphaLISA platform (PerkenElmer), as described previously [ 47 ]. Briefly, streptavidin modified donor beads were bound to biotin-labeled recombinant human PRG4 (Lubris BioPharma, Naples, FL) [ 48 ], and anti-PRG4 mAb 94D6 was bound to acceptor beads.…”

Endogenous production of hyaluronan, PRG4, and cytokines is sensitive to cyclic loading in synoviocytes

“…Conditioned media samples from 24 hours after the completion of loading were analyzed for PRG4 concentration using the AlphaLISA platform (PerkenElmer), as described previously [45]. Briefly, streptavidin modified donor beads were bound to biotin-labeled recombinant human PRG4 (Lubris BioPharma, Naples, FL) [46], and anti-PRG4 mAb 94D6 was bound to acceptor beads.…”