Quantitative measurement of lacritin in the tear fluid of patients with Dry Eye Disease.

Abstract

Purpose : The protein lacritin has emerged as an important tear fluid biomarker that is depleted in all forms of Dry Eye Disease (DED). This study employed a novel electrochemical sensor and the gold-standard Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) to quantitatively measure lacritin in clinical samples.

Methods : Tear fluid samples were collected from 60 patients with DED and 60 age-matched healthy controls with no history of ocular disease in accordance with the ethical guidelines and regulations established by the Institutional Review Board (IRB). Tear fluid was collected from each subject using 0.5 μL glass capillary tubes. Samples were stored at -80 °C until use. Samples from 30 patients and 30 healthy controls were analyzed using the Evosep LC-Bruker timsTOF Pro2 mass spectrometer. Parallel Reaction Monitoring (PRM)--based targeted mass spectrometry techniques were used to achieve a more precise quantitation of lacritin. Two tryptic peptides were added to the samples as stable isotopically labeled internal standards: (i) SILLTEQALAK (+8, 13C6, 15N2-Lys) and, (ii) QFIENGSEFAQK (+8,13C6, 15N2-Lys). To create the sensor, gold screen printed electrodes were functionalized with an anti-lacitrin antibody. The functionalization of the gold electrodes was achieved using a photochemical immobilization technique (PIT). Samples from 30 patients with DED and 30 healthy controls were analyzed using the electrochemical sensor.

Results : Lacritin was detected in all tear fluid samples analyzed with LC-MS/MS. Identified peptides were primarily from the C-terminus of the lacritin polymer with consistent protein coverage of 35-47%. The electrochemical sensor is capable of detecting lacritin concentrations in the range from 25 μg/mL to 0.25 ng/mL. Reproducible results were obtained with less than 5 mL of fluid collection.

Conclusions : Quantitative measurements of lacritin can be reproducibly measured in the tear fluid samples from patients with and without DED. LC-MS/MS provides a gold standard basis that can be used in laboratory settings and the electrochemical sensor provides a point-of-care platform that can be used in patient clinics.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.