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

Proteins in tears have an important role in eye healthand havebeen shown as a promising source of disease biomarkers. The goal ofthis study was to develop a robust, sensitive, and targeted methodfor profiling tear proteins to examine the variability within a groupof healthy volunteers over three days. Inter-individual and inter-dayvariabilities were examined to contribute to understanding the normalvariations in the tear proteome, as well as to establish which proteinsmay be better candidates as eventual biomarkers of specific diseases.Tear samples collected on Schirmer strips were subjected to bottom-upproteomics, and resulting peptides were analyzed using an optimizedtargeted method measuring 226 proteins by liquid chromatography-scheduledmultiple reaction monitoring. This method was developed using an in-housedatabase of identified proteins from tears compiled from high-resolutiondata-dependent liquid chromatography tandem mass spectrometry data.The measurement of unique peptide signals can help better understandthe dynamics of each of these proteins in tears. Some interestingtrends were seen in specific pathways or protein classes, includinghigher variabilities for those involved in glycolysis, glutathionemetabolism, and cytoskeleton proteins and lower variation for thoseinvolving the degradation of the extracellular matrix. The overallaim of this study was to contribute to the field of tear proteomicswith the development of a novel and targeted method that is highlyamenable to the clinical laboratory using high flow LC and commonlyused triple quadrupole mass spectrometry while ensuring that proteinquantitation was reported based on unique peptides for each proteinand robust peak areas with data normalization. These results reporton variabilities on over 200 proteins that are robustly detected intear samples from healthy volunteers with a simple sample preparationprocedure.