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.

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.

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%).

This study aimed to identify dysregulated proteins in Schirmer strip samples (ScS) from Sjögren's syndrome dry eye (SSDE) patients to uncover key biological processes using untargeted proteomics. The focus then shifted to two dysregulated proteins, PRDX6 and CXCL17, due to their roles in oxidative stress and mucosal immunity, with the goal of exploring their potential as therapeutic targets. Their involvement was further investigated in vitro and validated in a larger SSDE cohort, assessing their relationship with clinical signs. ScS from 12 SSDE patients and 6 healthy controls underwent untargeted proteomic analysis. PRDX6 and CXCL17 levels in ScS from 39 SSDE patients were quantified using ELISA. In vitro, human corneal epithelial cells (HCEc) were exposed to hyperosmolarity or IFN-γ, and PRDX6 and CXCL17 expression was assessed by RT-qPCR for gene expression and by ELISA and immunocytochemistry for protein expression. Untargeted proteomics identified 111 dysregulated proteins in SSDE, highlighting alterations in oxidative stress, cell metabolism, cytoskeleton organization, and programmed cell death. Targeted proteomics showed positive correlations between PRDX6 levels and TBUT/Schirmer tests, and negative correlations with OSDI/Oxford scores. CXCL17 levels negatively correlated with the Oxford score. In vitro, PRDX6 and CXCL17 expression increased under hyperosmotic or inflammatory stress, displaying inverse trends compared to ScS from SSDE patients. This study elucidates the biological processes driving epithelial cell alterations in SSDE, focusing on oxidative stress and mucosal homeostasis. It underscores the significant roles of PRDX6 and CXCL17 in these processes, suggesting their potential as biomarkers or therapeutic targets for SSDE.

Analyse protéomique des larmes et recherches de biomarqueurs pour la sécheresse oculaire: La sécheresse oculaire (SSO) est une maladie multifactorielle de la surface oculaire caractérisée par une instabilité du film lacrymal, une hyperosmolarité, une inflammation de la surface oculaire et des anomalies neurosensorielles. Sa prévalence varie de 5% à 50% selon la population et les critères diagnostiques utilisés, ce qui en fait l'une des maladies oculaires les plus fréquentes. Ses facteurs de risque sont nombreux (âge, sexe féminin, déséquilibre hormonal, exposition aux écrans/lumière, lentilles de contact, carence en vitamines, infections, médicaments, conservateurs des collyres, désinfectants ménagers et industriels, polluants atmosphériques, cosmétiques, chirurgie ophtalmique et maladies systémiques et auto-immunes). Elle a un impact important sur la qualité de vie, les performances visuelles et l’exécution des tâches quotidiennes au travail notamment, entraînant une augmentation des cas d'anxiété et de dépression. Le diagnostic de sécheresse oculaire repose sur les symptômes et l'examen clinique, mais la discordance entre signes et symptômes ainsi que l’intrication avec d'autres affections de la surface oculaire comme l’allergie, peut compliquer l’établissement d’un diagnostic précis. Actuellement, malgré de nombreuses études proposant différentes entités moléculaires d’intérêt, il n'existe toujours pas de biomarqueur validé pour cette maladie, ce qui entrave les capacités de dépistage, de diagnostic et le suivi de la réponse au traitement. La protéomique des larmes, parmi d'autres technologies omiques puissantes, est devenue un outil prometteur pour identifier les changements au niveau moléculaire et permettre d’envisager de potentiels biomarqueurs pour la SSO. Cependant, le manque d'études permettant une validation clinique à grande échelle et le manque de critères de diagnostic standardisés représentent de véritables défis. Cette étude s'est d'abord consacrée à l'amélioration de la préparation des échantillons et à l'optimisation des méthodes pré-analytiques analysant les effets d’agents tensioactifs sur le rendement des étapes d’extraction des protéines et améliorant l'identification des protéines en spectrométrie de masse en incluant les protéines intracellulaires prélevées grâce à une bandelette de Schirmer (ScS), couramment utilisée par les cliniciens pour recueiller et quantifier les larmes. Nous avons pu caractériser des prélèvements de larmes chez des sujets sains selon différentes parties de la ScS à l'aide du timsTOF Pro. Une vue holistique des principales protéines présentes à partir des composants cellulaires et solubles, a été réalisée. Une analyse séparée des deux parties de la bandelette a permis l'identification de protéines supplémentaires et les enzymes constituaient le plus grand groupe de protéines parmi les protéines identifiées. Nous avons contribué à la description et à la comparaison de plusieurs voies de signalisation associées à la surface oculaire impliquant le système immunitaire ou la glycosylation des protéines, par exemple. Puis nous avons réalisé une compilation d’un ensemble de données protéomiques issues de différentes études permettant l’identification de 1010 enzymes. Enfin, ce travail de thèse a identifié dans le protéome lacrymal des patients diverses nouvelles cibles d'intérêt impliquées dans le cytosquelette, le stress oxydant, l'apoptose, la protection cellulaire et l'inflammation. Nous avons sélectionné certaines cibles pour les étudier dans des modèles in vitro et dans une plus grande cohorte de patients, montrant des résultats prometteurs. Cependant, des recherches supplémentaires et une validation clinique plus importante restent nécessaires pour mieux comprendre cette pathologie complexe. En conclusion, la sécheresse oculaire est une maladie de surface oculaire, handicapante au quotidien, et dont l’incidence ne cesse de croître et pour laquelle le développement de biomarqueurs validés pour un diagnostic précis et un traitement efficace reste indispensable.