Fátima M. Santos

Vitreous humor in the pathologic scope: Insights from proteomic approaches

The vitreous humor (VH) is the largest component of the eye. It is a colorless, gelatinous, highly hydrated matrix that fills the posterior segment of the eye between the lens and retina in vertebrates. In VH, a diversity of proteins that can influence retinal physiology is present, including growth factors, hormones, proteins with transporter activity, and enzymes. More importantly, the protein composition of VH has been described as being altered in a number of disease states. Therefore, attempts aiming at establishing a map of VH proteins and detecting putative biomarkers for ocular illness or protein fluctuations with putative physiologic significance were conducted over the last two decades, using proteomic approaches. Proteomic strategies often involve gel‐based or LC techniques as sample fractioning approaches, subsequently coupled with MS procedures. This set of studies resulted in the proteomic characterization of a range of ocular disease samples, with particular incidence on diabetic retinopathy. However, practical therapeutic applications arising from these studies are scarce at the moment. A pertinent example of therapeutic targets arising from VH proteomics has emerged concerning vasoproliferative factors present in the vitreous, which should be involved in neovascularization and subsequent fibrovascular proliferation of the retina, in ocular disease context. Therefore, this review attempts to sum up the information acquired from the proteomic approaches to ocular disease conducted in VH samples, highlighting its clinical potential for disclosing ocular disease mechanisms and engendering pharmacological therapeutic treatments.

Trends in Protein-Based Biosensor Assemblies for Drug Screening and Pharmaceutical Kinetic Studies

The selection of natural and chemical compounds for potential applications in new pharmaceutical formulations constitutes a time-consuming procedure in drug screening. To overcome this issue, new devices called biosensors, have already demonstrated their versatility and capacity for routine clinical diagnosis. Designed to perform analytical analysis for the detection of a particular analyte, biosensors based on the coupling of proteins to amperometric and optical devices have shown the appropriate selectivity, sensibility and accuracy. During the last years, the exponential demand for pharmacokinetic studies in the early phases of drug development, along with the need of lower molecular weight detection, have led to new biosensor structure materials with innovative immobilization strategies. The result has been the development of smaller, more reproducible biosensors with lower detection limits, and with a drastic reduction in the required sample volumes. Therefore in order to describe the main achievements in biosensor fields, the present review has the main aim of summarizing the essential strategies used to generate these specific devices, that can provide, under physiological conditions, a credible molecule profile and assess specific pharmacokinetic parameters.

Recovery of biological active catechol-O-methyltransferase isoforms from Q-sepharose.

The development of new catechol-O-methyltransferase inhibitors has led to an improvement in the treatment of Parkinsons disease. However, despite the fact that the soluble isoform has been extensively investigated, few studies have been published concerning membrane isoform chromatographic recovery and bioactivity levels. In this work, chromatographic profiles of both catechol-O-methyltransferase isoforms were compared using quaternary amine as a ligand to evaluate its activity levels and recovery rates. Results show that both proteins required different conditions for adsorption; the soluble isoform adsorption was performed at low ionic strength, while the membrane isoform required increasing linear salt gradient. However, the application of 0.5% Triton X-100 promoted membrane isoform adsorption even at low ionic strength. Indeed, chromatographic conditions of both isoforms became similar when detergents were applied. The developed methods also appear to be highly effective in bioactivity recovery, presenting rates of 107% for soluble protein and 67 and 91% for membrane isoform without and with detergents, respectively. The chromatographic strategies with and without detergents resulted in a 4.3- and sevenfold purification, respectively, corresponding to specific activity values of 331 and 496 nmol/h/mg. Thus, the use of Q-sepharose as anion exchanger was effective in the recovery of both enzymes, which is a requirement for further kinetic and pharmacological trials.

An Improved HPLC Method for Quantification of Metanephrine with Coulometric Detection

A rapid and straightforward analytical method, based on the use of RP-HPLC with coulometric detection, was developed and validated for the quantification of metanephrine, an O-methylated product in catechol-Omethyltransferase enzymatic assays. The isocratic separation was achieved on a reverse column with a mobile phase consisting of 0.1 M sodium dihydrogen phosphate, 0.024 M citric acid monohydrate, 0.5 mM sodium octyl sulphate and 9% acetonitrile (%v/v). The method was found to be linear between 0.25 and 15 nmol/mL with a determination coefficient of 0.9997 for metanephrine. Intra-and interday precision and accuracy were in conformity with the criteria accepted in bioanalytical method validation and the LOD and LLOQ were 0.25 nmol/mL. The main focus of the developed method is the lower LLOQ achieved that can have important implications in laboratory research for COMT activity determinations, in particular for the methionine 108/158 variant obtained either from native or recombinant extracts. Another major advantage of the present method is the shorter run times on automated chromatographic systems that allow the analysis of several samples in a short time. In addition, metanephrine was stable in the samples for at least 24 h at room temperature, for at least 24 h in HPLC system injector and for at least three freeze/thaw cycles. The developed method demonstrated higher sensitivity, precision, accuracy, stability, and linearity when compared with the methods previously described. Finally, a catechol-O-methyltransferase

Trends in proteomic analysis of human vitreous humor samples

Proteomic analysis of human vitreous humor (VH) may elucidate the pathogenesis of retinal ocular diseases and may provide information for the development of potential therapeutic targets due to its pivotal location near lens and retina. The discovery of whole VH proteome involves a complex analysis of thousands of proteins simultaneously. Therefore, in proteomic studies the protein fractionation is important for reducing sample complexity, facilitating the access to the low‐abundant proteins, and recognizing them as biotargets for clinical research. Although several separation methods have been used, gel‐based proteomics are the most popular and versatile ones applied for global protein separation. However, chromatographic methods and its combination with other separation techniques are now beginning to be used as promising set‐ups for VH protein identification. This review attempts to offer an overview of the techniques currently used with VH, exploring its methodological demands, exposing its advantages, and helping the reader to plan future experiences. Moreover, this review shows the relevance of VH proteomic analysis as a tool for the study of the mechanisms underlying some ocular diseases and for the development of new therapeutic approaches.

Proteome analysis of vitreous humor in retinal detachment using two different flow-charts for protein fractionation

The deeper understanding of retinal detachment (RD) pathogenesis may improve the visual outcome after surgery. Given the main role of the vitreous in retinal eye diseases, two strategies were explored to identify its proteome in RD. Fractionation techniques such as anion exchange chromatography (IEX) and SDS-PAGE combined with MALDI-TOF/TOF analysis allowed to identify 127 proteins in vitreous of RD patients. From these proteins, 19 were identified using only the IEX fractionation strategy, and 117 using a bidimensional (IEX and SDS-PAGE) fractionation. Of these proteins, 68 had not yet been found in other vitreous proteomic studies. The fractionation with IEX and SDS-PAGE largely improved the number of identified proteins proving that it is crucial to combine several methodologies to cover vitreous proteome.

iTRAQ Quantitative Proteomic Analysis of Vitreous from Patients with Retinal Detachment

Rhegmatogenous retinal detachment (RRD) is a potentially blinding condition characterized by a physical separation between neurosensory retina and retinal pigment epithelium. Quantitative proteomics can help to understand the changes that occur at the cellular level during RRD, providing additional information about the molecular mechanisms underlying its pathogenesis. In the present study, iTRAQ labeling was combined with two-dimensional LC-ESI-MS/MS to find expression changes in the proteome of vitreous from patients with RRD when compared to control samples. A total of 150 proteins were found differentially expressed in the vitreous of patients with RRD, including 96 overexpressed and 54 underexpressed. Several overexpressed proteins, several such as glycolytic enzymes (fructose-bisphosphate aldolase A, gamma-enolase, and phosphoglycerate kinase 1), glucose transporters (GLUT-1), growth factors (metalloproteinase inhibitor 1), and serine protease inhibitors (plasminogen activator inhibitor 1) are regulated by HIF-1, which suggests that HIF-1 signaling pathway can be triggered in response to RRD. Also, the accumulation of photoreceptor proteins, including phosducin, rhodopsin, and s-arrestin, and vimentin in vitreous may indicate that photoreceptor degeneration occurs in RRD. Also, the accumulation of photoreceptor proteins, including phosducin, rhodopsin, and s-arrestin, and vimentin in vitreous may indicate that photoreceptor degeneration occurs in RRD. Nevertheless, the differentially expressed proteins found in this study suggest that different mechanisms are activated after RRD to promote the survival of retinal cells through complex cellular responses.