Purchase this article with an account.
A.C. Len, M.S. Baker, M.C. Gillies; Characterisation of Human Vitreous Humour: A Proteomic Approach . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5517.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Since the vitreous humour (VH) is directly apposed to the inner retina, examining protein profiles in samples of vitreous obtained either post mortem or during pars plana vitrectomy from patients with retinal disease may provide unique insights into the pathogenesis of a range of blinding macular diseases. We are conducting this project to characterise the protein profile of normal human vitreous and to test the hypothesis that it differs depending on age, sex and degree of post mortem delay.
VH samples were obtained from patients during a pars plana vitrectomy or from deceased individuals who have no history of diabetes. VH samples were prepared for proteome analysis by firstly dialysing then lyophilizing the samples. The proteins from the VH samples were then solubilized in a protein solubilization cocktail containing surfactants, reducing agents and chaotropes. Solubilized proteins were then separated using two–dimensional gel electrophoresis (2–DGE). The first dimension was focused using both passive and cup loading sample delivery and in the second dimension proteins were separated based on their molecular weight using SDS–PAGE. Abundant proteins were excised, digested with trypsin and analysed via matrix assisted light desorption/ionisation–mass spectrometry and peak lists generated were used to query online protein databases for identification. Serum albumin depletion was conducted on VH and proteins were separated using 2–DGE.
In comparison with normal human plasma, human vitreous’ proteome map differs significantly; however, like in human plasma serum albumin is the most abundant protein. The presence of serum albumin can hinder the display of low abundance proteins, to avoid this situation a sample pre–fractionation technique was employed to selectively remove this highly abundant protein species. Approximately 200% more proteins were displayed over a pI range of 4–7, a pI range in which the majority of VH proteins isoelectrically focus.
Human vitreous has a unique proteome profile. The removal of high abundance proteins, such as serum albumin, from VH associated with retinal diseases will facilitate the identification of critical low abundance proteins which it can reasonably be anticipated will provide unique insights into the pathogenesis of macular disease.
This PDF is available to Subscribers Only