Expanded biochemical analyses of human tear fluid: Polyvalent faces of the schirmer strip.

The tear film forms a protective barrier between the ocular surface and the external environment. Despite its small volume, recent advancements in preanalytical and analytical procedures have enabled its in-depth analysis using multiple approaches. However, the diversity of tear film collection methods and the lack of standardization in pre-analytical methods represent the main obstacles to reproducible results and comparison among different studies. In this study, we first improved the pre-analytical procedures for the extraction of various molecular entities from Schirmer strips (ScS). Subsequently, our investigation focused on analyzing the molecular variances that might occur between two primary tear collection methods: capillary tube (CT) and ScS. Additionally, we examined different parts of the ScS to underscore these variations, which could serve as crucial factors for developing a standardized, optimized protocol for sample processing. Our results show that the inclusion of surfactants in the extraction process enhanced both the yield of protein extraction and the number of proteins identified in ScS, by effectively lysing the cells and improving the solubility of several intracellular proteins. In addition to proteins, nucleic acids could also be recovered for gene expression analyses, particularly from the bulb region of the ScS which is placed in the cul-de-sac. Despite their diluted nature, extracts from ScS remain a suitable material for retrieving tear proteins such as IL-17A at levels as low as the fg/mL range, thanks to highly sensitive immunoassays. Collection methods can affect measured tear protein levels. Lactoferrin is found in higher percentages in capillary electrophoresis analysis of tears collected using ScS compared to tears collected by CT (39.6 ± 4.8% versus 31 ± 4.4%).

Profiling tear film enzymes reveals major metabolic pathways involved in the homeostasis of the ocular surface.

The ocular surface (OS) enzymes are of great interest due to their potential for novel ocular drug development. We aimed first to profile and classify the enzymes of the OS to describe major biological processes and pathways that are involved in the maintenance of homeostasis. Second, we aimed to compare the enzymatic profiles between the two most common tear collection methods, capillary tubes (CT) and Schirmer strips (ScS). A comprehensive tear proteomic dataset was generated by pooling all enzymes identified from nine tear proteomic analyses of healthy subjects using mass spectrometry. In these studies, tear fluid was collected using CT (n = 4), ScS (n = 4) or both collection methods (n = 1). Classification and functional analysis of the enzymes was performed using a combination of bioinformatic tools. The dataset generated identified 1010 enzymes. The most representative classes were hydrolases (EC 3) and transferases (EC 2). Phosphotransferases, esterases and peptidases were the most represented subclasses. A large portion of the identified enzymes was common to both collection methods (n = 499). More enzymes were specifically detected in the ScS-extracted proteome. The major pathways in which the identified enzymes participate are related to the immune system and protein, carbohydrate and lipid metabolism. Metabolic processes for nucleosides, cellular amides, sugars and sulfur compounds constituted the most enriched biological processes. Knowledge of these molecules highly susceptible to pharmacological manipulation might help to predict the metabolism of ophthalmic medications and develop novel prodrug strategies as well as new drug delivery systems. Combining such extensive knowledge of the OS enzymes with new analytical approaches and techniques might create new prospects for understanding, predicting and manipulating the metabolism of ocular pharmaceuticals. Our study reports new, essential data on OS enzymes while also comparing the enzyme profiles obtained via the two most popular methods of tear collection, capillary tubes and Schirmer strips.

Proteomic Analysis of Tears and Conjunctival Cells Collected with Schirmer Strips Using timsTOF Pro: Preanalytical Considerations.

This study aimed to investigate the human proteome profile of samples collected from whole (W) Schirmer strips (ScS) and their two parts-the bulb (B) and the rest of the strip (R)-with a comprehensive proteomic approach using a trapped ion mobility mass spectrometer, the timsTOF Pro. Eight ScS were collected from two healthy subjects at four different visits to be separated into three batches, i.e., 4W, 4B, and 4R. In total, 1582 proteins were identified in the W, B, and R batches. Among all identified proteins, binding proteins (43.4%) and those with catalytic activity (42.2%) constituted more than 80% of the molecular functions. The most represented biological processes were cellular processes (31.2%), metabolic processes (20.8%), and biological regulation (13.1%). Enzymes were the most represented protein class (41%), consisting mainly of hydrolases (47.5%), oxidoreductases (22.1%), and transferases (16.7%). The bulb (B), which is in contact with the conjunctiva, might collect both tear and cell proteins and therefore promote the identification of more proteins. Processing B and R separately before mass spectrometry (MS) analysis, combined with the high data acquisition speed and the addition of ion-mobility-based separation in the timsTOF Pro, can bring a new dimension to biomarker investigations of a limited sample such as tear fluid.