Abstract
BackgroundThere is a long-lasting need for non-invasive, more accurate diagnostic techniques when evaluating primary Sjögren’s syndrome (pSS) patients. Incorporation of additional diagnostics involving screening for disease-specific biomarkers in biological fluid is a promising concept that requires further investigation. In the current study we aimed to explore novel disease biomarkers in saliva and tears from pSS patients.MethodsLiquid chromatography-mass spectrometry (LC-MS) was performed on stimulated whole saliva and tears from 27 pSS patients and 32 healthy controls, and salivary and tear proteomic biomarker profiles were generated. LC-MS was also combined with size exclusion chromatography to isolate extracellular vesicles (EVs) from both fluids. Nanoparticle tracking analysis was conducted on joint fractions from the saliva and tears to determine size distribution and concentration of EVs. Further EV characterisation was performed by immunoaffinity capture of CD9-positive EVs using magnetic beads, detected by flow cytometry. The LC-MS data were analysed for quantitative differences between patient and control groups using Scaffold, and the proteins were further analysed using the Database for Annotation, Visualization and Integrated Discovery (DAVID), for gene ontology overrepresentation, and the Search Tool for the Retrieval of Interacting Genes/Proteins for protein-protein interaction network analysis.ResultsUpregulation of proteins involved in innate immunity (LCN2), cell signalling (CALM) and wound repair (GRN and CALML5) were detected in saliva in pSS. Saliva EVs also displayed biomarkers critical for activation of the innate immune system (SIRPA and LSP1) and adipocyte differentiation (APMAP). Tear analysis indicated overexpression of proteins involved in TNF-α signalling (CPNE1) and B cell survival (PRDX3). Moreover, neutrophil gelatinase-associated lipocalin was upregulated in saliva and tears in pSS. Consistently, DAVID analysis demonstrated pathways of the adaptive immune response in saliva, of cellular component assembly for saliva EVs, and of metabolism and protein folding in tears in pSS patients.ConclusionsLC-MS of saliva and tears from pSS patients, solely and in combination with size-exclusion chromatography allowed screening for possible novel biomarkers encompassing both salivary and lacrimal disease target organs. This approach could provide additional diagnostic accuracy in pSS, and could possibly also be applied for staging and monitoring the disease.
Highlights
There is a long-lasting need for non-invasive, more accurate diagnostic techniques when evaluating primary Sjögren’s syndrome patients
Workflow for the identification of proteins upregulated in patients with primary Sjögren’s syndrome (pSS) The proteome of saliva, tear fluid, and extracellular vesicles (EV) of both saliva and tear fluid from patients and controls were examined by digestion of the proteins with trypsin, analysis of the proteins by Liquid chromatography-mass spectrometry (LC-MS), identification of the proteins using Mascot database searches and further data analysis using Scaffold to find quantitative differences based on the t test applied on MS2 total ion currents
gene ontology (GO) overrepresentation analysis using DAVID indicated that cellular pathways for the upregulated proteins in the whole saliva from the pSS patient group, in comparison to unregulated proteins, included lymphocyte-mediated immunity, calcium ion binding and the neutrophin signalling pathway
Summary
There is a long-lasting need for non-invasive, more accurate diagnostic techniques when evaluating primary Sjögren’s syndrome (pSS) patients. The main classification criteria used today when diagnosing primary SS (pSS) are the American-European Consensus Group (AECG) criteria from 2002 [7], which rely on evaluating symptoms of ocular and oral dryness, assessing the secretory ability of the exocrine glands, screening for anti-Ro and anti-La autoantibodies, and evaluating biopsies of minor salivary glands for mononuclear cell infiltration [8]. This routine assessment of minor salivary gland tissue and histological focus scoring has been employed to describe salivary gland involvement in SS [9, 10]. This is a semi-quantitative, invasive technique useful for patients with glandular dysfunctions without autoantibody production [11]
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