Analyzing changes in tear proteins of Sjogren’s syndrome patients with timsTOF pro

AbstractPurposeTo investigate changes in the tear proteome of patients with Sjögren's syndrome (SSp) using a high‐resolution trapped ion mobility spectrometry (TIMS) powered with parallel accumulation–serial fragmentation (PASEF).MethodsTear samples were collected using Schirmer strips from healthy controls (HC) and SSp, then extracted in ammonium bicarbonate. The protein content of each sample was normalized to 250 ng. Proteomics analysis was performed by using TIMS coupled quadrupole time‐of‐flight (timsTOF Pro) followed by data processing using MaxQuant software for protein identification. Differentially expressed proteins were detected by a Limma statistical test with a false discovery rate (FDR)˂1%. Protein Gene Ontology classification was performed by using Panther.ResultsThe proteins identified decreased by 23.9% in SSp compared to HC. Off the proteins identified in SSp, 90% were common with HC while only 68% of the proteins identified in HC were detected in SSp. A total of 175 proteins were significantly modulated in SSp with a fold‐change ≥ 1.5 (p‐value ≤0.05). None of the proteins detected only in SSp was among significantly modulated proteins but 25 proteins identified only in HC were significantly modulated. Catalytic activity (46.9%) and binding proteins (38.5%) formed the major molecular function groups among significantly modulated proteins. Proteins that exhibit binding activity were mainly down‐regulated in SSp while proteins involved in apoptosis such as caspase‐3 and proteasome subunits were up‐regulated. Several oxidoreductase enzymes (18) such as sulfhydryl oxidase‐1, Glutathione peroxidase‐3 and lactoperoxidase have been down‐regulated. Actins, actin‐binding proteins, tubulins, microtubule‐binding proteins were decreased in SSp. These results highlight an alteration in cellular process and antioxidase activity.ConclusionsA large amount of proteins expressed differently in SSp that involve important biological processes and signalling pathways have been revealed. The improved spatial resolution, sensitivity, speed, and specificity of timsTOF Pro can dramatically increase the ability to fully investigate the molecular factors behind the pathophysiology of SS.