Targeted Workflow Investigating Variations in the Tear Proteome by Liquid Chromatography Tandem Mass Spectrometry

[Image: see text] Proteins in tears have an important role in eye health and have been shown as a promising source of disease biomarkers. The goal of this study was to develop a robust, sensitive, and targeted method for profiling tear proteins to examine the variability within a group of healthy volunteers over three days. Inter-individual and inter-day variabilities were examined to contribute to understanding the normal variations in the tear proteome, as well as to establish which proteins may be better candidates as eventual biomarkers of specific diseases. Tear samples collected on Schirmer strips were subjected to bottom-up proteomics, and resulting peptides were analyzed using an optimized targeted method measuring 226 proteins by liquid chromatography-scheduled multiple reaction monitoring. This method was developed using an in-house database of identified proteins from tears compiled from high-resolution data-dependent liquid chromatography tandem mass spectrometry data. The measurement of unique peptide signals can help better understand the dynamics of each of these proteins in tears. Some interesting trends were seen in specific pathways or protein classes, including higher variabilities for those involved in glycolysis, glutathione metabolism, and cytoskeleton proteins and lower variation for those involving the degradation of the extracellular matrix. The overall aim of this study was to contribute to the field of tear proteomics with the development of a novel and targeted method that is highly amenable to the clinical laboratory using high flow LC and commonly used triple quadrupole mass spectrometry while ensuring that protein quantitation was reported based on unique peptides for each protein and robust peak areas with data normalization. These results report on variabilities on over 200 proteins that are robustly detected in tear samples from healthy volunteers with a simple sample preparation procedure.