Characterization of human tear proteome in dry eye syndrome

Abstract

In gist, there are several major findings emerging from the current study. First, several important differential expression of proteins that were never explored extensively in the ocular system were identified. In the discovery study to distinguish the different DES subgroups, 79 potential protein biomarkers were identified. Amongst these proteins, 13 highly differentially expressed proteins were verified by targeted MS analysis. Hence, utilization of multiple tear biomarkers considering multiple biological functional characteristics (i.e. secretion deficiency, metabolic processes, immune response, inflammatory response and protease inhibition) will result in significant improvement of DES diagnostic accuracy. Among the differential proteins, PRR4, ZG16B, SCGB2A1, DMBT1, PROL1, LACRT, ALDH3A1, ENO1, TF, S100A8, S100A9, PEBP1 and ORM1 were verified to be significantly differentially expressed in abundance in DES subgroups, and thus, are highly regarded as potential biomarkers for the pathology. Second, the results of this study successfully demonstrated that the highly abundant lacrimal gland-specific PRR4 and ZG16B represent the major increased proteins in reflex tears for the first time. This finding highlights the significant regulation of these proteins in tears for the protection of the ocular surface, whereby its deficiency in tears may contribute towards the pathogenesis of DES. Third, PRR4 proteoforms were extensively characterized in healthy human tears employing the MS system, which resulted in identification of a new PRR4 isoform designated as PRR4-N3. In this study, designation of PRR4 isoforms was systematically simplified as PRR4-N1, PRR4-N2 and PRR4-N3. In addition, combinations of four types of PTMs which are methylation, acetylation, oxidation and pyroglutamate formation were identified for PRR4. The exact functions of the PRR4 and the existence of the variable isoforms and PTMs remain to be explored. To date, the lack of information about the characteristics and standardized designations for PRR4 isoforms has hindered complete understanding of the functions of PRR4 in tears. Therefore, the characteristic profiles of PRR4 elucidated in this study will contribute to the existing body of knowledge and open new avenues for in-depth functional investigation. Fourth, a rapid and robust targeted data acquisition approach of in-solution digestion based LC-ESI-MS/MS strategy was optimized and developed to relatively quantify proteins in individual tear samples. In view of the fact that tear volume is typically limited in a case study or a population-based study, the samples are pooled in most experiments and this will consequently hinder the detection and characterization of specific protein isoforms (e.g. PRR4-N1/N2/N3, CST4/CST1, IgA1/2 etc.). However, with the targeted approach employed, only small amounts of tear samples are required and this targeted MS strategy was instrumental for quantifying proteins that cannot be effectively detected by classical antibody-based methods. Additionally, this targeted MS approach is advantageous since multiple protein isoform-specific peptides could be precisely detected in a single MS run. This approach will enable elucidation of protein isoforms regulation, which might have important roles in the various biological processes and can potentially be used as biomarkers or therapeutic targets/ mediators. In conclusion, this study had identified the major protein biomarkers for specific DES subgroups and further characterized the intricate proteome regulation during reflex tearing, especially the potential role of PRR4 proteoforms, which may be the key player in the protection and maintenance of dynamic balance of the ocular surface. The outcomes of the identification and characterization of these protein biomarkers in this study, when extrapolated to clinical application, can provide invaluable hints on development of specific diagnostic tool for clinical tests and are of great importance for the prognostic usage for improved clinical management of the disease. It is important to highlight here that these protein biomarker panels could be potentially developed for topical protein therapy as effective medical treatments for DES. To date, there are no effective protein therapies available for DES due to the limited major biomarkers identified. The identification of the major deficient proteins in DES in this study, especially PRR4, could be applied as biotherapeutics. Developments of topical eye drops for DES with these recombinant proteins may physiologically rescue the ocular surface when a deficiency is detected. In succession, insights into the characterized PRR4 proteoforms regulations and PRR4-mediated protein interactions, which remain to be elucidated, are proposed to be explored for further understanding of their importance in DES pathogenesis, diagnosis and possible treatment options.