Conclusion
These findings demonstrate that proteomic analysis of human tear proteins can be performed without the need to pool samples, and that development of analytic workflows must consider factors that may affect outcomes in studies focused on diverse clinical samples.
Methods
Tears were collected by Schirmer strip following topical ocular anesthetic application then individually stored at -80 °C prior to processing for proteomics. Tear proteins were extracted from Schirmer strips, digested using suspension trapping spin columns (S-Trap), and labeled with high multiplicity tandem mass tags (TMT). Peptide digests were then extensively fractionated by two-dimensional chromatography and analyzed by mass spectrometry to identify and measure changes in protein abundance in each sample. Analysis of select samples was performed to test protocols and to compare the impact of clinically relevant parameters. To facilitate comparison of separate TMT experiments, common pool samples were included in each TMT instrument run and internal reference scaling (IRS) was performed.
Purpose
Human tears contain abundant, diverse sets of proteins that may serve as biomarkers of ocular surface health. There is a need for reproducible
Results
Differences in subsets of tear proteins were noted for: geographic site of tear collection, contact lens use, and differences in tear fluid volume among individuals.