Combined Targeted Analysis of Metabolites and Proteins in Tear Fluid With Regard to Clinical Applications

Purpose: To establish a robust workflow for combined mass spectrometry-based analysis of metabolites and proteins in tear fluid with regard to clinical applicability. Methods: Tear fluid was taken from 12 healthy volunteers at different time points using specially designed Schirmer strips. Following the liquid extraction of metabolites from standardized punches, the remaining material was processed for bottom-up proteomics. Targeted metabolite profiling was performed adapting a metabolomics kit, which targets 188 metabolites from four different analyte classes. Proteomics was performed of the identical samples targeting 15 tear proteins relevant to ocular health. Results: Sixty metabolites could be consistently determined in all tear samples (98 metabolites were detectable in average) covering acylcarnitines, amino acids, biogenic amines, and glycerophospholipids. Following normalization, the majority of metabolites exhibited intraindividual variances of less than 20%, both regarding different times of sampling, and the individual eye. The targeted analysis of tear proteins revealed a mean intraindividual variation of 23% for the three most abundant proteins. Even extreme differences in tear secretion rates resulted in interindividual variability below 30% for 65 metabolites and two proteins. Conclusions: The newly established workflow can be used for combined targeted detection of metabolites and proteins in one punch of a Schirmer strip in a clinical setting. Translational Relevance: Our data about intra- and interindividual as well as intereye variation provide a valuable basis for the design of clinical studies, and for the applicability of multiplexed omits to well accessible tear fluid with regard to future routine use.