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X. Gu, X. Yuan, J. S. Crabb, K. Shadrach, J. G. Hollyfield, J. W. Crabb; Quantitative Proteomic Analysis of AMD Bruch’s Membrane. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1753.
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Age-related changes in Bruch’s membrane lead to thickening and loss of permeability. We seek to identify and quantify proteome changes in Bruch’s membrane associated with age-related macular degeneration (AMD).
Samples of Bruch’s membrane (4-6 mm trephined discs) were isolated from the central region of human AMD and normal donor eyes . Protein was extracted in SDS, digested with trypsin, then peptides were labeled with diagnostic, amine specific, isobaric iTRAQ tags, disease and control digests combined and fractionated by strong cation exchange (SCX) chromatography. Alternatively, AMD and non-AMD Bruch’s membrane proteins were fractionated by SDS-PAGE in adjacent lanes, gel bands excised, digested in situ with trypsin, peptides labeled with iTRAQ tags, and gel band digests corresponding in apparent mass combined. iTRAQ-labled peptides were analyzed by on line LC MS/MS with a QTOF2 mass spectrometer and by off line LC MS/MS with a model 4800 MALDI TOF TOF mass spectrometer. Proteins were identified using the Mascot search engine and the Swiss-Protein sequence database. Relative protein quantification from iTRAQ labeling utilized a macro written in visual basic or GPS Explorer 3.6 software.
Relative protein quantitation has been obtained from Bruch’s membrane from 24 AMD donors individually compared with pooled, age-matched non-AMD donor samples. The LC MS/MS results from QTOF2 analyses and MALDI TOF/TOF analyses have been complementary and to-date have allowed quantification of over 900 proteins from two or more unique labeled peptides per protein. For proteins quantified in the majority of the AMD donors, the relative amount was greater for proteins such as vitronectin, alpha crystallin B, TIMP3, clusterin, and complement C3 and C9.
Quantitative mass spectrometric analysis offers promise for identifying AMD-associated and age-related changes in the protein composition of Bruch’s membrane. Current observations are consistent with previous analyses of Bruch’s membrane and drusen from AMD and normal donors (1999 IOVS 40, 2367; 2002 Proc. Natl. Acad. Sci. USA 99: 14682). More proteins were quantified following SCX chromatography of tryptic peptides than from SDS-PAGE prior to tryptic digestion. However, SDS-PAGE facilitated the preparation of detergent-solubilized samples, provided quantitation of different sized components of individual proteins, and yielded quantitative results comparable to that obtained with SCX fractionation
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