Method development for quantification of five tear proteins using selected reaction monitoring (SRM) mass spectrometry.

To establish the use of selected reaction monitoring (SRM) mass spectrometry for quantification of tear proteins. Tear samples were collected on multiple occasions (7-10 days) from healthy subjects with contact lens wear (CL = 3) and without contact lens wear (NCL = 4). Tear proteins were denatured using 8M urea, reduced with iodoacetamide, precipitated by acetone, and digested using trypsin. Internal standards were included by adding isotopically-labelled standards of known concentrations to the samples. Lactoferrin, lysozyme, prolactin-induced protein, lipocalin 1, and proline-rich protein 4 were quantified using liquid chromatography-triple quadruple mass spectrometry in conjunction with selected reaction monitoring. The limits of quantification for the selected peptides were below 50 pg/μL. The recovery of peptides from spiked digested tears was greater than or equal to 56% and the coefficient of variation values were less than or equal to 16%. The concentration of lactoferrin (1.20 ± 0.77 μg/μL), lysozyme (2.11 ± 1.50 μg/μL), and lipocalin-1 (1.75 ± 0.99 μg/μL) were consistent with previous ELISA studies. Tear levels of prolactin-induced protein (0.09 ± 0.06 μg/μL) and proline-rich 4 (0.80 ± 0.50 μg/μL) are reported here for the first time. The SRM method can be used for simultaneous detection and quantification of selected proteins in low volumes of human tear samples (2.5 μL per sample) without prior purification of each protein component or need for antibodies.