Javier Soria

Tear proteome and protein network analyses reveal a novel pentamarker panel for tear film characterization in dry eye and meibomian gland dysfunction.

Dry eye and meibomian gland dysfunction are common ocular surface disorders. Discrimination of both conditions often may be difficult given the overlapping of signs and symptoms, and the lack of correlation with clinical parameters. A total of 144 individuals were included in this study. To search for proteome differences, tear proteins were collected by Merocel sponge and analyzed using 2D-PAGE. Comparative tear protein profile analysis indicated changes in the expression levels of fifteen proteins. Subsequent to MALDI-TOF/TOF protein identification, network analysis revealed expression/interaction connections with other proteins, thereby identifying additional putative markers. A screening validation assay demonstrated the discriminative power of six candidate biomarkers. A further validation study using multiplexed-like ELISA assays in tear samples collected with both sponge and capillary confirmed the high discriminatory power of five biomarkers: S100A6, annexin A1 (ANXA1), annexin A11 (ANXA11), cystatin-S (CST4), and phospholipase A2-activating protein (PLAA) with an area under ROC curve (AUC)≥ 97.9% (sensitivity ≥ 94.3%; specificity ≥ 97.6%) when comparing dry eye and control individuals. This panel also discriminated between dry eye, meibomian gland dysfunction and control individuals, with a global correct assignment (CA) of 73.2% between all groups. Correct assignment was not found to be significantly dependent on the tear collection method.

Human Basal Tear Peptidome Characterization by CID, HCD, and ETD Followed by in Silico and in Vitro Analyses for Antimicrobial Peptide Identification.

Endogenous peptides are valuable targets in the analysis of biological processes. The tear film contains proteins and peptides released by the tear duct mucosal cells, including antimicrobial peptides involved in the protection against exogenous pathogens; however, the peptide content of the tear liquid remains poorly characterized. We analyzed naturally occurring peptides isolated from human basal tears. Mass spectrometry analysis of endogenous peptides presents a number of drawbacks, including size heterogeneity and nonpredictable fragmentation patterns, among others. Therefore, CID, ETD, and HCD methods were used for the characterization of the tear peptide content. The contribution of DMSO as an additive of the chromatographic solvents was also evaluated. We identified 157, 131, and 122 peptides using CID-, ETD-, and HCD-based methods, respectively. Altogether, 234 different peptides were identified, leading to the generation of the biggest data set of endogenous tear peptides to date. The antimicrobial activity prediction analysis performed in silico revealed different putative antimicrobial peptides. Two of the extracellular glycoprotein lacritin peptides were de novo synthesized, and their antimicrobial activity was confirmed in vitro. Our findings demonstrate the benefits of using different fragmentation methods for the analysis of endogenous peptides and provide a useful approach for the discovery of peptides with antimicrobial activity.

Human tear proteomics and peptidomics in ophthalmology: Toward the translation of proteomic biomarkers into clinical practice

Tears are a complex biological mixture containing electrolytes, metabolites, lipids, mucins, some small organic molecules, and proteins. The tear film has various roles in the lubrication, protection from the external environment, and nutrition of the cornea; it is also involved in the modulation of the optical properties of the eye. Tear composition reflects the physiological condition of the underlying tissues. Therefore, the tear fluid is useful in the evaluation of health and disease states and it is a valuable source of biomarkers for objective analysis of ocular and systemic diseases. The relatively high protein concentration of this fluid and the ease of noninvasive sample collection make it suitable for diagnostic and prognostic purposes. Efforts in proteomics research have positively affected to the field of ophthalmology, and the knowledge on the tear proteome has expanded considerably in the last few years. Nevertheless, despite a large amount of available data and the many biomarkers proposed for several eye and systemic diseases, the extent of translation to well-characterized and clinically useful tools has been largely insufficient. As for most of other biofluids, the road from discovery to clinical application is still long and full of pitfalls. In this review, we discuss the proteomic approaches used in the characterization of tear protein and peptide content, recapitulating the main studies and the progress done. We also present a brief summary of the path from discovery to clinical application of tear protein markers, with some representative examples of translation from the bench to the bedside. SIGNIFICANCE In this review we cover the most relevant proteomic approaches used in the characterization of the tear proteome, and for the first time we also focus in advances performed in the nowadays emerging peptide content characterization. In this context, we recapitulate on the main studies and the progresses done in this field. We also present a concise overview of the course that may be happen from discovery to clinical application for tear protein markers. Finally we include some representative examples of translation from the bench to the bedside.

Antigen-Antibody Affinity for Dry Eye Biomarkers by Label Free Biosensing. Comparison with the ELISA Technique

The specificity and affinity of antibody-antigen interactions is a fundamental way to achieve reliable biosensing responses. Different proteins involved with dry eye dysfunction: ANXA1, ANXA11, CST4, PRDX5, PLAA and S100A6; were validated as biomarkers. In this work several antibodies were tested for ANXA1, ANXA11 and PRDX5 to select the best candidates for each biomarker. The results were obtained by using Biophotonic Sensing Cells (BICELLs) as an efficient methodology for label-free biosensing and compared with the Enzyme-Linked Immuno Sorbent Assay (ELISA) technique.

Tear proteome analysis in ocular surface diseases using label-free LC-MS/MS and multiplexed-microarray biomarker validation

We analyzed the tear film proteome of patients with dry eye (DE), meibomian gland dysfunction (MGD), and normal volunteers (CT). Tear samples were collected from 70 individuals. Of these, 37 samples were analyzed using spectral-counting-based LC-MS/MS label-free quantitation, and 33 samples were evaluated in the validation of candidate biomarkers employing customized antibody microarray assays. Comparative analysis of tear protein profiles revealed differences in the expression levels of 26 proteins, including protein S100A6, annexin A1, cystatin-S, thioredoxin, phospholipase A2, antileukoproteinase, and lactoperoxidase. Antibody microarray validation of CST4, S100A6, and MMP9 confirmed the accuracy of previously reported ELISA assays, with an area under ROC curve (AUC) of 87.5%. Clinical endpoint analysis showed a good correlation between biomarker concentrations and clinical parameters. In conclusion, different sets of proteins differentiate between the groups. Apolipoprotein D, S100A6, S100A8, and ceruloplasmin discriminate best between the DE and CT groups. The differences between antileukoproteinase, phospholipase A2, and lactoperoxidase levels allow the distinction between MGD and DE, and the changes in the levels of annexin A1, clusterin, and alpha-1-acid glycoprotein 1, between MGD and CT groups. The functional network analysis revealed the main biological processes that should be examined to identify new candidate biomarkers and therapeutic targets.

The analysis of human conjunctival epithelium proteome in ocular surface diseases using impression cytology and 2D-DIGE

Abstract Conjunctival impression cytology samples from patients with meibomian gland dysfunction (MGD), dry eye (DE), and healthy subjects (CT) were collected for determination of the degree of squamous metaplasia (SM) by PAS‐hematoxylin staining and for comparative proteomic analyses by 2D‐DIGE. The protein spots with discriminant expression were identified by MALDI‐TOF/TOF mass spectrometry. Three independent statistical studies were conducted: i). Analysis of differential protein expression between study groups: We observed increased expression of proteins S100A4, S100A8, retinal dehydrogenase‐1, peroxiredoxin‐1, annexin‐A1, annexin‐A2, &agr;‐enolase, and glutathione S‐transferase‐P in DE, whereas the highest expression of peroxiredoxin‐6, actin cytoplasmic‐1, peroxiredoxin‐2, and heat shock protein HSP‐90‐&agr; was observed in MGD; ii). Correlation between changes in the proteome profile and the grade of SM: The expression of 5 different cytokeratins (KRT1, KRT4, KRT8, KRT10, and KRT13) correlated with the degree of SM; iii). Proteome profile differences between pathological and CT groups: An overall proteome analysis revealed upregulation of 9 proteins in the pathological groups (Annexin‐A1, &agr;‐enolase, Annexin‐A2, S100A8, cytokeratin‐1, Peroxiredoxin‐2 and Leukocyte elastase inhibitor) and downregulation of 2 proteins (Galectin‐3 and Lipocalin‐1). In conclusion, a sensitive proteomic approach to study conjunctival tissue collected from minimally invasive impression cytology was implemented. Differential proteomics analyses showed that in comparison with the MGD, the DE patients presented higher overexpression of proteins related to antimicrobial defense, tissue‐damage response, and regulation of body fluid secretions. Changes in MGD proteome were associated with oxidative stress and anti‐apoptotic processes. We found a correlation between the grade of SM and expression of proteins associated with cytoskeleton and keratinization. The studied pathological groups shared elements related to the defense and inflammatory responses. Dot blot assays of proteins ANXA1, S100A8, and S100A4 validated the proteomic results obtained from 2D‐DIGE experiments and confirmed the correlation between the expression of these proteins and the clinical parameters. Graphical abstract Figure. No caption available. HighlightsWe performed 2DE‐DIGE analysis of dry eye and MGD diseases in conjunctiva.We optimized conjunctival proteome analysis obtained by impression cytology.We present a panel of proteins able to differentiate between studied groups.Validation by dot‐blot assays of three proteins confirmed 2DE‐DIGE results.

Custom RT-qPCR-array for glaucoma filtering surgery prognosis

Excessive subconjunctival scarring is the main reason of failure of glaucoma filtration surgery. We analyzed conjunctival and systemic gene expression patterns after non penetrating deep sclerectomy (NPDS). To find expression patterns related to surgical failure and their correlation with the clinical outcomes. This study consisted of two consecutive stages. The first was a prospective analysis of wound-healing gene expression profile of six patients after NPDS. Conjunctival samples and peripheral blood samples were collected before and 15, 90,180, and 360 days after surgery. In the second stage, we conducted a retrospective analysis correlating the late conjunctival gene expression and the outcome of the NPDS for 11 patients. We developed a RT-qPCR Array for 88 key genes associated to wound healing. RT-qPCR Array analysis of conjunctiva samples showed statistically significant differences in 29/88 genes in the early stages after surgery, 20/88 genes between 90 and 180 days after surgery, and only 2/88 genes one year after surgery. In the blood samples, the most important changes occurred in 12/88 genes in the first 15 days after surgery. Correspondence analyses (COA) revealed significant differences between the expression of 20/88 genes in patients with surgical success and failure one year after surgery. Different expression patterns of mediators of the bleb wound healing were identified. Examination of such patterns might be used in surgery prognosis. RT-qPCR Array provides a powerful tool for investigation of differential gene expression wound healing after glaucoma surgery.