Abstract
Mapping of proteins involved in normal eye functions is a prerequisite to identify pathological changes during eye disease processes. We therefore analysed the proteome of human vitreous by applying in-depth proteomic screening technologies. For ethical reasons human vitreous samples were obtained by vitrectomy from “surrogate normal patients” with epiretinal gliosis that is considered to constitute only negligible pathological vitreoretinal changes. We applied different protein prefractionation strategies including liquid phase isoelectric focussing, 1D SDS gel electrophoresis and a combination of both and compared the number of identified proteins obtained by the respective method. Liquid phase isoelectric focussing followed by SDS gel electrophoresis increased the number of identified proteins by a factor of five compared to the analysis of crude unseparated human vitreous. Depending on the prefractionation method proteins were subjected to trypsin digestion either in-gel or in solution and the resulting peptides were analysed on a UPLC system coupled online to an LTQ Orbitrap XL mass spectrometer. The obtained mass spectra were searched against the SwissProt database using the Mascot search engine. Bioinformatics tools were used to annotate known biological functions to the detected proteins. Following this strategy we examined the vitreous proteomes of three individuals and identified 1111 unique proteins. Besides structural, transport and binding proteins, we detected 261 proteins with known enzymatic activity, 51 proteases, 35 protease inhibitors, 35 members of complement and coagulation cascades, 15 peptide hormones, 5 growth factors, 11 cytokines, 47 receptors, 30 proteins of visual perception, 91 proteins involved in apoptosis regulation and 265 proteins with signalling activity. This highly complex mixture strikingly differs from the human plasma proteome. Thus human vitreous fluid seems to be a unique body fluid. 262 unique proteins were detected which are present in all three patient samples indicating that these might represent the constitutive protein pattern of human vitreous. The presented catalogue of human vitreous proteins will enhance our understanding of physiological processes in the eye and provides the groundwork for future studies on pathological vitreous proteome changes.
Highlights
The human vitreous humor is a clear aqueous solution which fills the posterior compartment of the eye, located between the lens and the retina
In order to test whether additional prefractionation of the samples by liquid phase isolelectric focussing prior to SDS gel electrophoresis may enable digging deeper into the vitreous proteome, sample VP2 was subjected to such prefractionation procedure which reduces sample complexity by resolving the proteome into 5 fractions according to the proteins’ isoelectric points
On the other hand, when liquid phase IEF was applied without subsequent SDS-gel electrophoresis only 284 proteins were detectable in VP2, among these were 17 additional proteins not detected in the standard procedure
Summary
The human vitreous humor is a clear aqueous solution which fills the posterior compartment of the eye, located between the lens and the retina. With aging the vitreous undergoes a process of progressive liquefaction so that at the age of 80–90 years more function of soluble extracellular proteins is maintenance of homeostasis in adjacent tissues, vitreous fluid contains soluble proteins that are necessary to sustain normal ocular morphology and functions. Such important physiological role requires tight control of the qualitative and quantitative composition of the vitreous proteome. We are still far from a detailed understanding of the complex biochemical processes mediated by vitreous proteins, and extensive analysis of the role of vitreous proteome changes in the development of eye diseases is not yet feasible
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